Fungal resistant plants expressing ACD

ABSTRACT

The present invention relates to a method of increasing resistance against fungal pathogens of the family Phacosporaceae in plants and/or plant cells. This is achieved by increasing the expression of an ACD protein or fragment thereof in a plant, plant part and/or plant cell in comparison to wild type plants, wild type plant parts and/or wild type plant cells. Furthermore, the invention relates to transgenic plants, plant parts, and/or plant cells having an increased resistance against fungal pathogens, in particular, pathogens of the family Phacopsoraceae, and to recombinant expression vectors comprising a sequence that is identical or homologous to a sequence encoding an ACD protein.

This application is a National Stage application of InternationalApplication No. PCT/EP2013/055347, filed Mar. 15, 2013, which claims thebenefit of U.S. Provisional Application No. 61/620,452, filed Apr. 5,2012. This application also claims priority under 35 U.S.C. §119 toEuropean Patent Application No. 12163265.7, filed Apr. 5, 2012.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

This application was filed electronically via EFS-Web and includes anelectronically submitted sequence listing in .txt format. The .txt filecontains a sequence listing entitled “Sequence_List.txt” created on Mar.31, 2014, and is 53,248 bytes in size. The sequence listing contained inthis .txt file is part of the specification and is hereby incorporatedby reference herein in its entirety.

SUMMARY OF THE INVENTION

The present invention relates to a method of increasing resistanceagainst fungal pathogens, in particular, pathogens of the familyPhacopsoraceae, for example soybean rust, in plants, plant parts, and/orplant cells. This is achieved by increasing the expression and/oractivity of a ACD protein in a plant, plant part and/or plant cell incomparison to wild type plants, wild type plant parts and/or wild typeplant cells.

Furthermore, the invention relates to transgenic plants, plant parts,and/or plant cells having an increased resistance against fungalpathogens, in particular, pathogens of the family Phacopsoraceae, forexample soybean rust, and to recombinant expression vectors comprising asequence that is identical or homologous to a sequence encoding a ACDprotein.

BACKGROUND OF THE INVENTION

The cultivation of agricultural crop plants serves mainly for theproduction of foodstuffs for humans and animals. Monocultures inparticular, which are the rule nowadays, are highly susceptible to anepidemic-like spreading of diseases. The result is markedly reducedyields. To date, the pathogenic organisms have been controlled mainly byusing pesticides. Nowadays, the possibility of directly modifying thegenetic disposition of a plant or pathogen is also open to man.

Resistance generally describes the ability of a plant to prevent, or atleast curtail the infestation and colonization by a harmful pathogen.Different mechanisms can be discerned in the naturally occurringresistance, with which the plants fend off colonization byphytopathogenic organisms. These specific interactions between thepathogen and the host determine the course of infection (Schopfer andBrennicke (1999) Pflanzenphysiologie, Springer Verlag,Berlin-Heidelberg, Germany).

With regard to the race specific resistance, also called hostresistance, a differentiation is made between compatible andincompatible interactions. In the compatible interaction, an interactionoccurs between a virulent pathogen and a susceptible plant. The pathogensurvives, and may build up reproduction structures, while the hostmostly dies off. An incompatible interaction occurs on the other handwhen the pathogen infects the plant but is inhibited in its growthbefore or after weak development of symptoms. In the latter case, theplant is resistant to the respective pathogen (Schopfer and Brennicke,vide supra). However, this type of resistance is specific for a certainstrain or pathogen.

In both compatible and incompatible interactions a defensive andspecific reaction of the host to the pathogen occurs. In nature,however, this resistance is often overcome because of the rapidevolutionary development of new virulent races of the pathogens (Neu etal. (2003) American Cytopathol. Society, MPMI 16 No. 7: 626-633).

Most pathogens are plant-species specific. This means that a pathogencan induce a disease in a certain plant species, but not in other plantspecies (Heath (2002) Can. J. Plant Pathol. 24: 259-264). The resistanceagainst a pathogen in certain plant species is called non-hostresistance. The non-host resistance offers strong, broad, and permanentprotection from phytopathogens. Genes providing non-host resistanceprovide the opportunity of a strong, broad and permanent protectionagainst certain diseases in non-host plants. In particular, such aresistance works for different strains of the pathogen.

Fungi are distributed worldwide. Approximately 100 000 different fungalspecies are known to date. Thereof rusts are of great importance. Theycan have a complicated development cycle with up to five different sporestages (spermatium, aecidiospore, uredospore, teleutospore andbasidiospore).

During the infection of plants by pathogenic fungi, different phases areusually observed. The first phases of the interaction betweenphytopathogenic fungi and their potential host plants are decisive forthe colonization of the plant by the fungus. During the first stage ofthe infection, the spores become attached to the surface of the plants,germinate, and the fungus penetrates the plant. Fungi may penetrate theplant via existing ports such as stomata, lenticels, hydatodes andwounds, or else they penetrate the plant epidermis directly as theresult of the mechanical force and with the aid of cell-wall-digestingenzymes. Specific infection structures are developed for penetration ofthe plant. The soybean rust Phakopsora pachyrhizi directly penetratesthe plant epidermis. After crossing the epidermal cell, the fungusreaches the intercellular space of the mesophyll, where the fungusstarts to spread through the leaves. To acquire nutrients the funguspenetrates mesophyll cells and develops haustoria inside the mesophyllcell. During the penetration process the plasmamembrane of thepenetrated mesophyll cell stays intact. Therefore the soybean rustfungus establishes a biotrophic interaction with soybean.

The biotrophic phytopathogenic fungi, such as many rusts, depend fortheir nutrition on the metabolism of living cells of the plants. Thistype of fungi belong to the group of biotrophic fungi, like other rustfungi, powdery mildew fungi or oomycete pathogens like the genusPhytophthora or Peronospora. The necrotrophic phytopathogenic fungidepend for their nutrition on dead cells of the plants, e.g. speciesfrom the genus Fusarium, Rhizoctonia or Mycospaerella. Soybean rust hasoccupied an intermediate position, since it penetrates the epidermisdirectly, whereupon the penetrated cell becomes necrotic. After thepenetration, the fungus changes over to an obligatory-biotrophiclifestyle. The subgroup of the biotrophic fungal pathogens which followsessentially such an infection strategy is heminecrotrohic. In contrastto a heminecrotrophic pathogen, a hemibiotrophic pathogen lives for ashort period of time in a biotrophic manner and subsequently startskilling the host cell and/or host organism, i.e., changes for the restof its life-cycle to a necrotrophic life-style.

Soybean rust has become increasingly important in recent times. Thedisease may be caused by the biotrophic rusts Phakopsora pachyrhizi(Sydow) and Phakopsora meibomiae (Arthur). They belong to the classBasidiomycota, order Uredinales, family Phakopsoraceae. Both rustsinfect a wide spectrum of leguminosic host plants. P. pachyrhizi, alsoreferred to as Asian rust, is the more aggressive pathogen on soy(Glycine max), and is therefore, at least currently, of great importancefor agriculture. P. pachyrhizi can be found in nearly all tropical andsubtropical soy growing regions of the world. P. pachyrhizi is capableof infecting 31 species from 17 families of the Leguminosae undernatural conditions and is capable of growing on further 60 species undercontrolled conditions (Sinclair et al. (eds.), Proceedings of the rustworkshop (1995), National SoyaResearch Laboratory, Publication No. 1(1996); Rytter J. L. et al., Plant Dis. 87, 818 (1984)). P. meibomiaehas been found in the Caribbean Basin and in Puerto Rico, and has notcaused substantial damage as yet.

P. pachyrhizi can currently be controlled in the field only by means offungicides. Soy plants with resistance to the entire spectrum of theisolates are not available. When searching for resistant plants, sixdominant genes Rpp1-5 and Rpp? (Hyuuga), which mediate resistance of soyto P. pachyrhizi, were discovered. The resistance was lost rapidly, asP. pychyrhizi develops new virulent races.

In recent years, fungal diseases, e.g. soybean rust, has gained inimportance as pest in agricultural production. There was therefore ademand in the prior art for developing methods to control fungi and toprovide fungal resistant plants.

Much research has been performed on the field of powdery and downymildew infecting the epidermal layer of plants. However, the problem tocope with soybean rust which infects the mesophyll remains unsolved.

The object of the present invention is inter alia to provide a method ofincreasing resistance against fungal pathogens, preferably rustpathogens (i.e., fungal pathogens of the order Pucciniales), preferablyagainst fungal pathogens of the family Phacopsoraceae, more preferablyagainst fungal pathogens of the genus Phacopsora, most preferablyagainst Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur),also known as soybean rust.

Surprisingly, we found that fungal pathogens, in particular rustpathogens (i.e., fungal pathogens of the order Pucciniales), preferablyfungal pathogens of the family Phacopsoraceae, for example soybean rust,can be controlled by overexpression of the ethylene precursor degradingenzyme aminocyclopropane carboxylic acid deaminase (ACD). Thus, withoutbeing limited by theory, we found that fungal resistance can be achievedby inhibition of the ethylene signaling pathway, at least byoverexpression of an ACD gene, in a plant, plant part, and/or plantcell.

The present invention therefore provides a method of increasingresistance against fungal pathogens, preferably against rust pathogens(i.e., fungal pathogens of the order Pucciniales), preferably againstfungal pathogens of the family Phacopsoraceae, more preferably againstfungal pathogens of the genus Phacopsora, most preferably againstPhakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur), alsoknown as soybean rust, in transgenic plants, transgenic plant parts, ortransgenic plant cells by overexpressing one or more ACD nucleic acids.

A further object is to provide transgenic plants resistant against rustpathogens (i.e., fungal pathogens of the order Pucciniales), preferablyagainst fungal pathogens of the family Phacopsoraceae, more preferablyagainst fungal pathogens of the genus Phacopsora, most preferablyagainst Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur),also known as soybean rust, a method for producing such plants as wellas a vector construct useful for the above methods.

Therefore, the present invention also refers to a recombinant vectorconstruct and a transgenic plant, transgenic plant part, or transgenicplant cell comprising an exogenous ACD nucleic acid. Furthermore, amethod for the production of a transgenic plant, transgenic plant partor transgenic plant cell using the nucleic acid of the present inventionis claimed herein. In addition, the use of a nucleic acid or therecombinant vector of the present invention for the transformation of aplant, plant part, or plant cell is claimed herein.

The objects of the present invention, as outlined above, are achieved bythe subject-matter of the main claims. Preferred embodiments of theinvention are defined by the subject matter of the dependent claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the examples included herein.

DEFINITIONS

Unless otherwise noted, the terms used herein are to be understoodaccording to conventional usage by those of ordinary skill in therelevant art. In addition to the definitions of terms provided herein,definitions of common terms in molecular biology may also be found inRieger et al., 1991 Glossary of genetics: classical and molecular, 5thEd., Berlin: Springer-Verlag; and in Current Protocols in MolecularBiology, F. M. Ausubel et al., Eds., Current Protocols, a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(1998 Supplement).

It is to be understood that as used in the specification and in theclaims, “a” or “an” can mean one or more, depending upon the context inwhich it is used. Thus, for example, reference to “a cell” can mean thatat least one cell can be utilized. It is to be understood that theterminology used herein is for the purpose of describing specificembodiments only and is not intended to be limiting.

Throughout this application, various publications are referenced. Thedisclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains. Standard techniquesfor cloning, DNA isolation, amplification and purification, forenzymatic reactions involving DNA ligase, DNA polymerase, restrictionendonucleases and the like, and various separation techniques are thoseknown and commonly employed by those skilled in the art. A number ofstandard techniques are described in Sambrook et al., 1989 MolecularCloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y.;Maniatis et al., 1982 Molecular Cloning, Cold Spring Harbor Laboratory,Plainview, N.Y.; Wu (Ed.) 1993 Meth. Enzymol. 218, Part I; Wu (Ed.) 1979Meth Enzymol. 68; Wu et al., (Eds.) 1983 Meth. Enzymol. 100 and 101;Grossman and Moldave (Eds.) 1980 Meth. Enzymol. 65; Miller (Ed.) 1972Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.; Old and Primrose, 1981 Principles of GeneManipulation, University of California Press, Berkeley; Schleif andWensink, 1982 Practical Methods in Molecular Biology; Glover (Ed.) 1985DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins(Eds.) 1985 Nucleic Acid Hybridization, IRL Press, Oxford, UK; andSetlow and Hollaender 1979 Genetic Engineering: Principles and Methods,Vols. 1-4, Plenum Press, New York. Abbreviations and nomenclature, whereemployed, are deemed standard in the field and commonly used inprofessional journals such as those cited herein.

The terms “inhibition of the ethylene signaling pathway”, “reduction ofthe ethylene signaling pathway”, or “suppression of the ethylenepathway” or “inactivation of the ethylene pathway” means that theethylene signaling pathway, e.g., as shown in FIG. 1, in a plant, plantpart, or plant cell is disturbed as compared to a wildtype plant, plantpart, or plant cell. Preferably, the disturbance of the ethylenesignaling pathway leads to a reduced rate of ethylene production, a lossof ethylene production or a lowered ethylene content as compared to awildtype plant, plant part, or plant cell exposed to the sameconditions. Furthermore, the ethylene signaling pathway can be disturbedby altering the activity of one or more ethylene signaling compoundswith or without effecting the ethylene production or ethylene content.

“Homologues” of a protein encompass peptides, oligopeptides,polypeptides, proteins and/or enzymes having amino acid substitutions,deletions and/or insertions relative to the unmodified protein inquestion and having similar functional activity as the unmodifiedprotein from which they are derived.

“Homologues” of a nucleic acid encompass nucleotides and/orpolynucleotides having nucleic acid substitutions, deletions and/orinsertions relative to the unmodified nucleic acid in question, whereinthe protein coded by such nucleic acids has similar or higher functionalactivity as the unmodified protein coded by the unmodified nucleic acidfrom which they are derived. In particular, homologues of a nucleic acidmay encompass substitutions on the basis of the degenerative amino acidcode.

A “deletion” refers to removal of one or more amino acids from a proteinor to the removal of one or more nucleic acids from DNA, ssRNA and/ordsRNA.

An “insertion” refers to one or more amino acid residues or nucleic acidresidues being introduced into a predetermined site in a protein or thenucleic acid.

A “substitution” refers to replacement of amino acids of the proteinwith other amino acids having similar properties (such as similarhydrophobicity, hydrophilicity, antigenicity, propensity to form orbreak α-helical structures or beta-sheet structures).

On the nucleic acid level a substitution refers to a replacement ofnucleic acid with other nucleic acids, wherein the protein coded by themodified nucleic acid has a similar function. In particular homologuesof a nucleic acid encompass substitutions on the basis of thedegenerative amino acid code.

Amino acid substitutions are typically of single residues, but may beclustered depending upon functional constraints placed upon the proteinand may range from 1 to 10 amino acids; insertions or deletion willusually be of the order of about 1 to 10 amino acid residues. The aminoacid substitutions are preferably conservative amino acid substitutions.Conservative substitution tables are well known in the art (see forexample Creighton (1984) Proteins. W.H. Freeman and Company (Eds) andTable 1 below).

TABLE 1 Examples of conserved amino acid substitutions ConservativeConservative Residue Substitutions Residue Substitutions Ala Ser LeuIle; Val Arg Lys Lys Arg; Gln Asn Gln; His Met Leu; Ile Asp Glu Phe Met;Leu; Tyr Gln Asn Ser Thr; Gly Cys Ser Thr Ser; Val Glu Asp Trp Tyr GlyPro Tyr Trp; Phe His Asn; Gln Val Ile; Leu Ile Leu, Val

Amino acid substitutions, deletions and/or insertions may readily bemade using peptide synthetic techniques well known in the art, such assolid phase peptide synthesis and the like, or by recombinant DNAmanipulation.

Methods for the manipulation of DNA sequences to produce substitution,insertion or deletion variants of a protein are well known in the art.For example, techniques for making substitution mutations atpredetermined sites in DNA are well known to those skilled in the artand include M13 mutagenesis, T7-Gene in vitro mutagenesis (USB,Cleveland, Ohio), QuickChange Site Directed mutagenesis (Stratagene, SanDiego, Calif.), PCR-mediated site-directed mutagenesis or othersite-directed mutagenesis protocols.

Orthologues and paralogues encompass evolutionary concepts used todescribe the ancestral relationships of genes. Paralogues are geneswithin the same species that have originated through duplication of anancestral gene; orthologues are genes from different organisms that haveoriginated through speciation, and are also derived from a commonancestral gene.

The term “domain” refers to a set of amino acids conserved at specificpositions along an alignment of sequences of evolutionarily relatedproteins. While amino acids at other positions can vary betweenhomologues, amino acids that are highly conserved at specific positionsindicate amino acids that are likely essential in the structure,stability or function of a protein.

Specialist databases exist for the identification of domains, forexample, SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95,5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244),InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318), Prosite(Bucher and Bairoch (1994), A generalized profile syntax forbiomolecular sequences motifs and its function in automatic sequenceinterpretation. (In) ISMB-94; Proceedings 2nd International Conferenceon Intelligent Systems for Molecular Biology. Altman R., Brutlag D.,Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAI Press, Menlo Park;Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)), or Pfam (Batemanet al., Nucleic Acids Research 30(1): 276-280 (2002)). A set of toolsfor in silico analysis of protein sequences is available on the ExPASyproteomics server (Swiss Institute of Bioinformatics (Gasteiger et al.,ExPASy: the proteomics server for in-depth protein knowledge andanalysis, Nucleic Acids Res. 31:3784-3788 (2003)). Domains or motifs mayalso be identified using routine techniques, such as by sequencealignment.

Methods for the alignment of sequences for comparison are well known inthe art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAPuses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48:443-453) to find the global (i.e. spanning the complete sequences)alignment of two sequences that maximizes the number of matches andminimizes the number of gaps. The BLAST algorithm (Altschul et al.(1990) J Mol Biol 215: 403-10) calculates percent sequence identity orsimilarity or homology and performs a statistical analysis of theidentity or similarity or homology between the two sequences. Thesoftware for performing BLAST analysis is publicly available through theNational Centre for Biotechnology Information (NCBI). Homologues mayreadily be identified using, for example, the ClustalW multiple sequencealignment algorithm (version 1.83), with the default pairwise alignmentparameters, and a scoring method in percentage. Global percentages ofsimilarity/homology/identity may also be determined using one of themethods available in the MatGAT software package (Campanella et al., BMCBioinformatics. 2003 Jul. 10; 4:29. MatGAT: an application thatgenerates similarity/homology/identity matrices using protein or DNAsequences.). Minor manual editing may be performed to optimise alignmentbetween conserved motifs, as would be apparent to a person skilled inthe art. Furthermore, instead of using full-length sequences for theidentification of homologues, specific domains may also be used. Thesequence identity values may be determined over the entire nucleic acidor amino acid sequence or over selected domains or conserved motif(s),using the programs mentioned above using the default parameters. Forlocal alignments, the Smith-Waterman algorithm is particularly useful(Smith T F, Waterman M S (1981) J. Mol. Biol 147(1); 195-7).

As used herein the terms “fungal-resistance”, “resistant to a fungus”and/or “fungal-resistant” mean reducing, preventing, or delaying aninfection by fungi. The term “resistance” refers to fungal resistance.Resistance does not imply that the plant necessarily has 100% resistanceto infection. In preferred embodiments, enhancing or increasing fungalresistance means that resistance in a resistant plant is greater than10%, greater than 20%, greater than 30%, greater than 40%, greater than50%, greater than 60%, greater than 70%, greater than 80%, greater than90%, or greater than 95% in comparison to a wild type plant.

As used herein the terms “soybean rust-resistance”, “resistant to asoybean rust”, “soybean rust-resistant”, “rust-resistance”, “resistantto a rust”, or “rust-resistant” mean reducing or preventing or delayingan infection of a plant, plant part, or plant cell by Phacopsoracea, inparticular Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae(Arthur)—also known as soybean rust or Asian Soybean Rust (ASR), ascompared to a wild type plant, wild type plant part, or wild type plantcell. Resistance does not imply that the plant necessarily has 100%resistance to infection. In preferred embodiments, enhancing orincreasing rust resistance means that rust resistance in a resistantplant is greater than 10%, greater than 20%, greater than 30%, greaterthan 40%, greater than 50%, greater than 60%, greater than 70%, greaterthan 80%, greater than 90%, or greater than 95% in comparison to a wildtype plant that is not resistant to soybean rust. Preferably the wildtype plant is a plant of a similar, more preferably identical, genotypeas the plant having increased resistance to the soybean rust, but doesnot comprise an exogenous ACD nucleic acid, functional fragments thereofand/or an exogenous nucleic acid capable of hybridizing with an ACDnucleic acid.

The level of fungal resistance of a plant can be determined in variousways, e.g. by scoring/measuring the infected leaf area in relation tothe overall leaf area. Another possibility to determine the level ofresistance is to count the number of soybean rust colonies on the plantor to measure the amount of spores produced by these colonies. Anotherway to resolve the degree of fungal infestation is to specificallymeasure the amount of rust DNA by quantitative (q) PCR. Specific probesand primer sequences for most fungal pathogens are available in theliterature (Frederick R D, Snyder C L, Peterson G L, et al. 2002Polymerase chain reaction assays for the detection and discrimination ofthe rust pathogens Phakopsora pachyrhizi and P. meibomiae,Phytopathology 92(2) 217-227).

The term “hybridization” as used herein includes “any process by which astrand of nucleic acid molecule joins with a complementary strandthrough base pairing” (J. Coombs (1994) Dictionary of Biotechnology,Stockton Press, New York). Hybridization and the strength ofhybridization (i.e., the strength of the association between the nucleicacid molecules) is impacted by such factors as the degree ofcomplementarity between the nucleic acid molecules, stringency of theconditions involved, the Tm of the formed hybrid, and the G:C ratiowithin the nucleic acid molecules.

As used herein, the term “Tm” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the Tm ofnucleic acid molecules is well known in the art. As indicated bystandard references, a simple estimate of the Tm value may be calculatedby the equation: Tm=81.5+0.41(% G+C), when a nucleic acid molecule is inaqueous solution at 1 M NaCl (see e.g., Anderson and Young, QuantitativeFilter Hybridization, in Nucleic Acid Hybridization (1985). Otherreferences include more sophisticated computations, which takestructural as well as sequence characteristics into account for thecalculation of Tm. Stringent conditions, are known to those skilled inthe art and can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

In particular, the term “stringency conditions” refers to conditions,wherein 100 contigous nucleotides or more, 150 contigous nucleotides ormore, 200 contigous nucleotides or more or 250 contigous nucleotides ormore which are a fragment or identical to the complementary nucleic acidmolecule (DNA, RNA, ssDNA or ssRNA) hybridizes under conditionsequivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 MNaPO4, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C. or65° C., preferably at 65° C., with a specific nucleic acid molecule(DNA; RNA, ssDNA or ss RNA). Preferably, the hybridizing conditions areequivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 MNaPO4, 1 mM EDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C. or65° C., preferably 65° C., more preferably the hybridizing conditionsare equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 50° C.or 65° C., preferably 65° C. Preferably, the complementary nucleotideshybridize with a fragment or the whole ACD nucleic acids. Preferably,the complementary polynucleotide hybridizes with parts of the ACDnucleic acids capable to provide soybean rust resistance byoverexpression or downregulation, respectively.

“Identity” or “homology” or “similarity” between two nucleic acidssequences or amino acid sequences refers in each case over the entirelength of the ACD nucleic acid sequences or ACD amino acid sequences.The terms “identity”, “homology” and “similarity” are used hereininterchangeably.

For example the identity may be calculated by means of the Vector NTISuite 7.1 program of the company Informax (USA) employing the ClustalMethod (Higgins D G, Sharp P M. Fast and sensitive multiple sequencealignments on a microcomputer. Comput Appl. Biosci. 1989 April;5(2):151-1) with the following settings:

Multiple Alignment Parameter:

Gap opening penalty 10 Gap extension penalty 10 Gap separation penaltyrange  8 Gap separation penalty off % identity for alignment delay 40Residue specific gaps off Hydrophilic residue gap off Transitionweighing  0

Pairwise Alignment Parameter:

FAST algorithm on K-tuple size 1 Gap penalty 3 Window size 5 Number ofbest diagonals 5

Alternatively the identity may be determined according to Chenna, Ramu,Sugawara, Hideaki, Koike, Tadashi, Lopez, Rodrigo, Gibson, Toby J,Higgins, Desmond G, Thompson, Julie D. Multiple sequence alignment withthe Clustal series of programs. (2003) Nucleic Acids Res 31(13):3497-500, the web page:http://www.ebi.ac.uk/Tools/clustalw/index.html# and the followingsettings

DNA Gap Open Penalty 15.0 DNA Gap Extension Penalty 6.66 DNA MatrixIdentity Protein Gap Open Penalty 10.0 Protein Gap Extension Penalty 0.2Protein matrix Gonnet Protein/DNA ENDGAP −1 Protein/DNA GAPDIST 4

All the nucleic acid sequences mentioned herein (single-stranded anddouble-stranded DNA and RNA sequences, for example cDNA and mRNA) can beproduced in a known way by chemical synthesis from the nucleotidebuilding blocks, e.g. by fragment condensation of individualoverlapping, complementary nucleic acid building blocks of the doublehelix. Chemical synthesis of oligonucleotides can, for example, beperformed in a known way, by the phosphoamidite method (Voet, Voet, 2ndedition, Wiley Press, New York, pages 896-897). The accumulation ofsynthetic oligonucleotides and filling of gaps by means of the Klenowfragment of DNA polymerase and ligation reactions as well as generalcloning techniques are described in Sambrook et al. (1989), see below.

Sequence identity between the nucleic acid or protein useful accordingto the present invention and the ACD nucleic acids or ACD proteins maybe optimized by sequence comparison and alignment algorithms known inthe art (see Gribskov and Devereux, Sequence Analysis Primer, StocktonPress, 1991, and references cited therein) and calculating the percentdifference between the nucleotide or protein sequences by, for example,the Smith-Waterman algorithm as implemented in the BESTFIT softwareprogram using default parameters (e.g., University of Wisconsin GeneticComputing Group).

The term “plant” is intended to encompass plants at any stage ofmaturity or development, as well as any tissues or organs (plant parts)taken or derived from any such plant unless otherwise clearly indicatedby context. Plant parts include, but are not limited to, plant cells,stems, roots, flowers, ovules, stamens, seeds, leaves, embryos,meristematic regions, callus tissue, anther cultures, gametophytes,sporophytes, pollen, microspores, protoplasts, hairy root cultures,and/or the like. The present invention also includes seeds produced bythe plants of the present invention. Preferably, the seeds comprise theexogenous ACD nucleic acids. In one embodiment, the seeds can developinto plants with increased resistance to fungal infection as compared toa wild-type variety of the plant seed. As used herein, a “plant cell”includes, but is not limited to, a protoplast, gamete producing cell,and a cell that regenerates into a whole plant. Tissue culture ofvarious tissues of plants and regeneration of plants therefrom is wellknown in the art and is widely published.

Reference herein to an “endogenous” nucleic acid and/or protein refersto the nucleic acid and/or protein in question as found in a plant inits natural form (i.e., without there being any human intervention).

The term “exogenous” nucleic acid refers to a nucleic acid that has beenintroduced in a plant by means of genetechnology. An “exogenous” nucleicacid can either not occur in a plant in its natural form, be differentfrom the nucleic acid in question as found in a plant in its naturalform, or can be identical to a nucleic acid found in a plant in itsnatural form, but integrated not within their natural geneticenvironment. The corresponding meaning of “exogenous” is applied in thecontext of protein expression. For example, a transgenic plantcontaining a transgene, i.e., an exogenous nucleic acid, may, whencompared to the expression of the endogenous gene, encounter asubstantial increase of the expression of the respective gene or proteinin total. A transgenic plant according to the present invention includesan exogenous ACD nucleic acid integrated at any genetic loci andoptionally the plant may also include the endogenous gene within thenatural genetic background.

For the purposes of the invention, “recombinant” means with regard to,for example, a nucleic acid sequence, a nucleic acid molecule, anexpression cassette or a vector construct comprising any one or more ACDnucleic acids, all those constructions brought about by man bygentechnological methods in which either

-   (a) the sequences of the ACD nucleic acids or a part thereof, or-   (b) genetic control sequence(s) which is operably linked with the    ACD nucleic acid sequence according to the invention, for example a    promoter, or-   (c) a) and b)    are not located in their natural genetic environment or have been    modified by man by gentechnological methods. The modification may    take the form of, for example, a substitution, addition, deletion,    inversion or insertion of one or more nucleotide residues. The    natural genetic environment is understood as meaning the natural    genomic or chromosomal locus in the original plant or the presence    in a genomic library or the combination with the natural promoter.

A recombinant nucleic acid may also refer to a nucleic acid in anisolated form. A recombinant nucleic acid, expression cassette or vectorconstruct preferably comprises a natural gene and a natural promoter, anatural gene and a non-natural promoter, a non-natural gene and anatural promoter, or a non-natural gene and a non-natural promoter.

In the case of a genomic library, the natural genetic environment of thenucleic acid sequence is preferably retained, at least in part. Theenvironment flanks the nucleic acid sequence at least on one side andhas a sequence length of at least 50 bp, preferably at least 500 bp,especially preferably at least 1000 bp, most preferably at least 5000bp.

A naturally occurring expression cassette—for example the naturallyoccurring combination of the natural promoter of the nucleic acidsequences with the corresponding nucleic acid sequence encoding aprotein useful in the methods of the present invention, as definedabove—becomes a recombinant expression cassette when this expressioncassette is modified by man by non-natural, synthetic (“artificial”)methods such as, for example, mutagenic treatment. Suitable methods aredescribed, for example, in U.S. Pat. No. 5,565,350, WO 00/15815 orUS200405323. Furthermore, a naturally occurring expression cassette—forexample the naturally occurring combination of the natural promoter ofthe nucleic acid sequences with the corresponding nucleic acid sequenceencoding a protein useful in the methods of the present invention, asdefined above—becomes a recombinant expression cassette when thisexpression cassette is not integrated in the natural genetic environmentbut in a different genetic environment.

It shall further be noted that in the context of the present invention,the term “isolated nucleic acid” or “isolated protein” may in someinstances be considered as a synonym for a “recombinant nucleic acid” ora “recombinant protein”, respectively and refers to a nucleic acid orprotein that is not located in its natural genetic environment and/orthat has been modified by genetechnical methods. The isolated gene maybe isolated from an organism or may be manmade, for example by chemicalsynthesis.

As used herein, the term “transgenic” refers to an organism, e.g., aplant, plant cell, callus, plant tissue, or plant part that exogenouslycontains the nucleic acid, recombinant construct, vector or expressioncassette described herein or a part thereof which is preferablyintroduced by non-essentially biological processes, preferably byAgrobacteria transformation. The recombinant construct or a part thereofis stably integrated into a chromosome, so that it is passed on tosuccessive generations by clonal propagation, vegetative propagation orsexual propagation. Preferred successive generations are transgenic too.Essentially biological processes may be crossing of plants and/ornatural recombination.

A transgenic plant, plants cell or tissue for the purposes of theinvention is thus understood as meaning that an exogenous ACD nucleicacid, recombinant construct, vector or expression cassette including oneor more ACD nucleic acids is integrated into the genome by means ofgenetechnology.

Preferably, constructs or vectors or expression cassettes are notpresent in the genome of the original plant or are present in the genomeof the transgenic plant not at their natural locus of the genome of theoriginal plant.

A “wild type” plant, “wild type” plant part, or “wild type” plant cellmeans that said plant, plant part, or plant cell does not expressexogenous ACD nucleic acid or exogenous ACD protein.

Natural locus means the location on a specific chromosome, preferablythe location between certain genes, more preferably the same sequencebackground as in the original plant which is transformed.

Preferably, the transgenic plant, plant cell or tissue thereof expressesthe ACD nucleic acids, ACD constructs or ACD expression cassettesdescribed herein.

The term “expression” or “gene expression” means the transcription of aspecific gene or specific genes or specific genetic vector construct.The term “expression” or “gene expression” in particular means thetranscription of a gene or genes or genetic vector construct intostructural RNA (rRNA, tRNA), or mRNA with or without subsequenttranslation of the latter into a protein. The process includestranscription of DNA and processing of the resulting RNA product. Theterm “expression” or “gene expression” can also include the translationof the mRNA and therewith the synthesis of the encoded protein, i.e.,protein expression.

The term “increased expression” or “enhanced expression” or“overexpression” or “increase of content” as used herein means any formof expression that is additional to the original wild-type expressionlevel. For the purposes of this invention, the original wild-typeexpression level might also be zero (absence of expression).

Methods for increasing expression of genes or gene products are welldocumented in the art and include, for example, overexpression driven byappropriate promoters, the use of transcription enhancers or translationenhancers. Isolated nucleic acids which serve as promoter or enhancerelements may be introduced in an appropriate position (typicallyupstream) of a non-heterologous form of a polynucleotide so as toupregulate expression of a nucleic acid encoding the protein ofinterest. For example, endogenous promoters may be altered in vivo bymutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No.5,565,350; Zarling et al., WO9322443), or isolated promoters may beintroduced into a plant cell in the proper orientation and distance froma gene of the present invention so as to control the expression of thegene.

If protein expression is desired, it is generally desirable to include apolyadenylation region at the 3′-end of a polynucleotide coding region.The polyadenylation region can be derived from the natural gene, from avariety of other plant genes, or from T-DNA. The 3′ end sequence to beadded may be derived from, for example, the nopaline synthase oroctopine synthase genes, or alternatively from another plant gene, orless preferably from any other eukaryotic gene.

An intron sequence may also be added to the 5′ untranslated region (UTR)and/or the coding sequence of the partial coding sequence to increasethe amount of the mature message that accumulates in the cytosol.Inclusion of a spliceable intron in the transcription unit in both plantand animal expression constructs has been shown to increase geneexpression at both the mRNA and protein levels up to 1000-fold (Buchmanand Berg (1988) Mol. Cell biol. 8: 4395-4405; Callis et al. (1987) GenesDev 1:1183-1200). Such intron enhancement of gene expression istypically greatest when placed near the 5′ end of the transcriptionunit. Use of the maize introns Adh1-S intron 1, 2, and 6, the Bronze-1intron are known in the art. For general information see: The MaizeHandbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).

The term “functional fragment” refers to any nucleic acid or proteinwhich comprises merely a part of the full length nucleic acid or fulllength protein, respectively, but still provides the same function,e.g., fungal resistance, when expressed or repressed in a plant,respectively. Preferably, the fragment comprises at least 50%, at least60%, at least 70%, at least 80%, at least 90% at least 95%, at least98%, at least 99% of the original sequence. Preferably, the functionalfragment comprises contiguous nucleic acids or amino acids as in theoriginal nucleic acid or original protein, respectively. In oneembodiment the fragment of any of the ACD nucleic acids has an identityas defined above over a length of at least 20%, at least 30%, at least50%, at least 75%, at least 90% of the nucleotides of the respective ACDnucleic acid.

In cases where overexpression of nucleic acid is desired, the term“similar functional activity” or “similar function” means that anyhomologue and/or fragment provide fungal resistance when expressed in aplant. Preferably similar functional activity means at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 98%, at least 99% or 100% or higher fungal resistance comparedwith functional activity provided by the exogenous expression of the ACDnucleotide sequence as defined by SEQ ID NO: 1 or the ACD proteinsequence as defined by SEQ ID NO: 2.

The term “increased activity” or “enhanced activity” as used hereinmeans any protein having increased activity and which provides anincreased fungal resistance compared with the wildtype plant merelyexpressing the respective endogenous ACD nucleic acid. As far asoverexpression is concerned, for the purposes of this invention, theoriginal wild-type expression level might also be zero (absence ofexpression).

With respect to a vector construct and/or the recombinant nucleic acidmolecules, the term “operatively linked” is intended to mean that thenucleic acid to be expressed is linked to the regulatory sequence,including promoters, terminators, enhancers and/or other expressioncontrol elements (e.g., polyadenylation signals), in a manner whichallows for expression of the nucleic acid (e.g., in a host plant cellwhen the vector is introduced into the host plant cell). Such regulatorysequences are described, for example, in Goeddel, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990) and Gruber and Crosby, in: Methods in Plant Molecular Biology andBiotechnology, Eds. Glick and Thompson, Chapter 7, 89-108, CRC Press:Boca Raton, Fla., including the references therein. Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cells and those that direct expression ofthe nucleotide sequence only in certain host cells or under certainconditions. It will be appreciated by those skilled in the art that thedesign of the vector can depend on such factors as the choice of thehost cell to be transformed, the level of expression of nucleic aciddesired, and the like.

The term “introduction” or “transformation” as referred to hereinencompass the transfer of an exogenous polynucleotide into a host cell,irrespective of the method used for transfer. Plant tissue capable ofsubsequent clonal propagation, whether by organogenesis orembryogenesis, may be transformed with a vector construct of the presentinvention and a whole plant regenerated there from. The particulartissue chosen will vary depending on the clonal propagation systemsavailable for, and best suited to, the particular species beingtransformed. Exemplary tissue targets include leaf disks, pollen,embryos, cotyledons, hypocotyls, megagametophytes, callus tissue,existing meristematic tissue (e.g., apical meristem, axillary buds, androot meristems), and induced meristem tissue (e.g., cotyledon meristemand hypocotyl meristem). The polynucleotide may be transiently or stablyintroduced into a host cell and may be maintained non-integrated, forexample, as a plasmid. Alternatively, it may be integrated into the hostgenome. The host genome includes the nucleic acid contained in thenucleus as well as the nucleic acid contained in the plastids, e.g.,chloroplasts, and/or mitochondria. The resulting transformed plant cellmay then be used to regenerate a transformed plant in a manner known topersons skilled in the art.

The term “terminator” encompasses a control sequence which is a DNAsequence at the end of a transcriptional unit which signals 3′processing and polyadenylation of a primary transcript and terminationof transcription. The terminator can be derived from the natural gene,from a variety of other plant genes, or from T-DNA. The terminator to beadded may be derived from, for example, the nopaline synthase oroctopine synthase genes, or alternatively from another plant gene, orless preferably from any other eukaryotic gene.

DETAILED DESCRIPTION

The ACD nucleic acid to be overexpressed in order to achieve increasedresistance to fungal pathogens, e.g., of the family Phacopsoraceae, forexample soybean rust, is preferably a nucleic acid coding for anaminocyclopropane carboxylic acid deaminase (ACD) protein, and ispreferably as defined by SEQ ID NO: 1, 3-10, 11, 13, 15, 17, 19, 21, 23,or 25, or a fragment, homolog, derivative, orthologue or paraloguethereof. Preferably, the nucleic acid coding for an aminocyclopropanecarboxylic acid deaminase (ACD) protein of the present invention has atleast 60% identity, preferably at least 70% sequence identity, at least80%, at least 90%, at least 95%, at least 98%, at least 99% sequenceidentity, or even 100% sequence identity with SEQ ID NO: 1, 3-10, 11,13, 15, 17, 19, 21, 23, or 25 or is a functional fragment thereof. Mostpreferred is at least 95% identity, more preferred is at least 98% or atleast 99% identity. Percentages of identity of a nucleic acid areindicated with reference to the entire nucleotide region given in asequence specifically disclosed herein. Preferably, the ACD nucleic acidcomprises at least about 100, at least about 200, at least about 300, atleast about 400, at least about 500, at least about 600, at least about700, at least about 800, at least about 850, at least about 900, atleast about 950, at least about 975, at least about 990, at least about1000, or at least about 1010 nucleotides, preferably continuousnucleotides, preferably counted from the 5′ or 3′ end of the nucleicacid or up to the full length of the nucleic acid sequence set out inSEQ ID NO: 1, 3-10, 11, 13, 15, 17, 19, 21, 23, or 25.

Preferably, the nucleic acid coding for an aminocyclopropane carboxylicacid deaminase (ACD) protein of the present invention has at least 60%identity, preferably at least 70% sequence identity, at least 80%, atleast 90%, at least 95%, at least 98%, at least 99% sequence identity,or even 100% sequence identity with SEQ ID NO: 1 or is a functionalfragment thereof. Most preferred is at least 95% identity, morepreferred is at least 98% or at least 99% identity. Percentages ofidentity of a nucleic acid are indicated with reference to the entirenucleotide region given in a sequence specifically disclosed herein.Preferably, the ACD nucleic acid comprises at least about 100, at leastabout 200, at least about 300, at least about 400, at least about 500,at least about 600, at least about 700, at least about 800, at leastabout 850, at least about 900, at least about 950, at least about 975,at least about 990, at least about 1000, or at least about 1010nucleotides, preferably continuous nucleotides, preferably counted fromthe 5′ or 3′ end of the nucleic acid or up to the full length of thenucleic acid sequence set out in SEQ ID NO: 1.

The ACD protein preferably is a 1-aminocyclopropane-1-carboxylic aciddeaminase, and preferably defined by SEQ ID NO: 2, 12, 14, 16, 18, 20,22, 24, or 26, or a fragment, homolog, derivative, orthologue orparalogue thereof. Preferably, the ACD protein of the present inventionis encoded by a nucleic acid, which has at least 60% identity,preferably at least 70% sequence identity, at least 80%, at least 90%,at least 95%, at least 98%, at least 99% sequence identity, or even 100%sequence identity with SEQ ID NO: 1, 3-10, 11, 13, 15, 17, 19, 21, 23,or 25 or a functional fragment thereof. More preferably, the ACD proteinof the present invention has at least 60%, preferably at least 70%sequence identity, at least 80%, at least 90%, at least 95%, at least98%, at least 99% sequence identity, or even 100% sequence identity withSEQ ID NO: 2, 12, 14, 16, 18, 20, 22, 24, or 26, or is a functionalfragment thereof, an orthologue or a paralogue thereof. Most preferredis at least 95% identity, more preferred is at least 98% or at least 99%identity. Percentages of identity of a polypeptide or protein areindicated with reference to the entire amino acid sequence specificallydisclosed herein. Preferably, the ACD protein comprises at least about50, at least about 75, at least about 100, at least about 125, at leastabout 150, at least about 175, at least about 200, at least about 225,at least about 250, at least about 275, at least about 300, at leastabout 310, at least about 320, at least about 325, at least about 330 orat least about 335 amino acid residues, preferably continuous amino acidresidues, preferably counted from the N-terminus or the C-terminus ofthe amino acid sequence, or up to the full length of the amino acidsequence set out in SEQ ID NO: 2, 12, 14, 16, 18, 20, 22, 24, or 26.

The ACD protein preferably is a 1-aminocyclopropane-1-carboxylic aciddeaminase, and preferably defined by SEQ ID NO: 2, or a fragment,homolog, derivative, orthologue or paralogue thereof. Preferably, theACD protein of the present invention is encoded by a nucleic acid, whichhas at least 60% identity, preferably at least 70% sequence identity, atleast 80%, at least 90%, at least 95%, at least 98%, at least 99%sequence identity, or even 100% sequence identity with SEQ ID NO: 1 or afunctional fragment thereof. More preferably, the ACD protein of thepresent invention has at least 60%, preferably at least 70% sequenceidentity, at least 80%, at least 90%, at least 95%, at least 98%, atleast 99% sequence identity, or even 100% sequence identity with SEQ IDNO: 2, or is a functional fragment thereof, an orthologue or a paraloguethereof. Most preferred is at least 95% identity, more preferred is atleast 98% or at least 99% identity. Percentages of identity of apolypeptide or protein are indicated with reference to the entire aminoacid sequence specifically disclosed herein. Preferably, the ACD proteincomprises at least about 50, at least about 75, at least about 100, atleast about 125, at least about 150, at least about 175, at least about200, at least about 225, at least about 250, at least about 275, atleast about 300, at least about 310, at least about 320, at least about325, at least about 330 or at least about 335 amino acid residues,preferably continuous amino acid residues, preferably counted from theN-terminus or the C-terminus of the amino acid sequence, or up to thefull length of the amino acid sequence set out in SEQ ID NO: 2.

One embodiment of the invention is a method for increasing fungalresistance, preferably resistance to Phacopsoracea, for example soy beanrust, in a plant, plant part, or plant cell by increasing the expressionof a ACD protein or a functional fragment, orthologue, paralogue orhomologue thereof in comparison to wild-type plants, wild-type plantparts or wild-type plant cells.

The present invention also provides a method for increasing resistanceto fungal pathogens, in particular fungal pathogens of the familyPhacopsoraceae, preferably against fungal pathogens of the genusPhacopsora, most preferably against Phakopsora pachyrhizi (Sydow) andPhakopsora meibomiae (Arthur), also known as soy bean rust in plants orplant cells, wherein in comparison to wild type plants, wild type plantparts, or wild type plant cells a ACD protein is overexpressed.

The present invention further provides a method for increasingresistance to fungal pathogens of the genus Phacopsora, most preferablyagainst Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur),also known as soy bean rust in plants or plant cells by overexpressionof a ACD protein.

In preferred embodiments, the protein amount and/or function of the ACDprotein in the plant is increased by at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or at least 95% or more in comparison to a wildtype plant that is not transformed with the ACD nucleic acid.

In one embodiment of the invention, the ACD protein is encoded by

-   (i) a nucleic acid having at least 60%, preferably at least 70%, for    example at least 75%, more preferably at least 80%, for example at    least 85%, even more preferably at least 90%, for example at least    95% or at least 96% or at least 97% or at least 98% most preferably    99% identity with SEQ ID NO: 1, 3-10, 11, 13, 15, 17, 19, 21, 23, or    25, a functional fragment thereof, or an orthologue or a paralogue    thereof; or by-   (ii) a nucleic acid encoding a protein having at least 60% identity,    preferably at least 70%, for example at least 75%, more preferably    at least 80%, for example at least 85%, even more preferably at    least 90%, for example at least 95% or at least 96% or at least 97%    or at least 98% most preferably 99% homology with SEQ ID NO: 2, 12,    14, 16, 18, 20, 22, 24, or 26, a functional fragment thereof, an    orthologue or a paralogue thereof, preferably the ACD nucleic acid    encodes a ACD protein that has essentially the same biological    activity as an ACD protein encoded by SEQ ID NO: 2; preferably the    encoded ACD protein confers enhanced fungal resistance relative to    control plants;-   (iii) a nucleic acid capable of hybridizing under stringent    conditions with any of the nucleic acids according to (i) or (ii) or    a complementary sequence (complement) thereof, and which preferably    encodes a ACD protein that has essentially the same biological    activity as an ACD protein encoded by SEQ ID NO: 2; preferably the    encoded ACD protein confers enhanced fungal resistance relative to    control plants; or by-   (iv) a nucleic acid encoding the same ACD protein as the ACD nucleic    acids of (i) to (iii) above, but differing from the ACD nucleic    acids of (i) to (iii) above due to the degeneracy of the genetic    code.

In one embodiment of the invention, the ACD protein is encoded by

-   (i) a nucleic acid having at least 60%, preferably at least 70%, for    example at least 75%, more preferably at least 80%, for example at    least 85%, even more preferably at least 90%, for example at least    95% or at least 96% or at least 97% or at least 98% most preferably    99% identity with SEQ ID NO: 1, a functional fragment thereof, or an    orthologue or a paralogue thereof; preferably the ACD protein that    has essentially the same biological activity as an ACD protein    encoded by SEQ ID NO: 2; preferably the ACD protein confers enhanced    fungal resistance relative to control plants; or by-   (ii) a nucleic acid encoding a protein having at least 60% identity,    preferably at least 70%, for example at least 75%, more preferably    at least 80%, for example at least 85%, even more preferably at    least 90%, for example at least 95% or at least 96% or at least 97%    or at least 98% most preferably 99% homology with SEQ ID NO: 2, a    functional fragment thereof, an orthologue or a paralogue thereof,    preferably the nucleic acid encodes a ACD protein that has    essentially the same biological activity as an ACD protein encoded    by SEQ ID NO: 2; preferably the encoded ACD protein confers enhanced    fungal resistance relative to control plants;-   (iii) a nucleic acid capable of hybridizing under stringent    conditions with any of the nucleic acids according to (i) or (ii) or    a complementary sequence (complement) thereof, and which preferably    encodes a ACD protein that has essentially the same biological    activity as an ACD protein encoded by SEQ ID NO: 2; preferably the    encoded ACD protein confers enhanced fungal resistance relative to    control plants; or by-   (iv) a nucleic acid encoding the same ACD protein as the ACD nucleic    acids of (i) to (iii) above, but differing from the ACD nucleic    acids of (i) to (iii) above due to the degeneracy of the genetic    code.

Another preferred embodiment is a method for increasing fungalresistance, preferably resistance to Phacopsoracea, for example soy beanrust, in a plant, plant part, or plant cell, by increasing theexpression of a ACD protein or a functional fragment, orthologue,paralogue or homologue thereof wherein the ACD protein is encoded by

-   (i) an exogenous nucleic acid having at least 60% identity,    preferably at least 70% sequence identity, at least 80%, at least    90%, at least 95%, at least 98%, at least 99% sequence identity, or    even 100% sequence identity with SEQ ID NO: 1, 3-10, 11, 13, 15, 17,    19, 21, 23, or 25 or a functional fragment thereof, an orthologue or    a paralogue thereof;-   (ii) an exogenous nucleic acid encoding a protein having at least    60%, preferably at least 70% sequence identity, at least 80%, at    least 90%, at least 95%, at least 98%, at least 99% sequence    identity, or even 100% sequence identity with SEQ ID NO: 2, a    functional fragment thereof, an orthologue or a paralogue thereof;-   (iii) an exogenous nucleic acid capable of hybridizing under    stringent conditions with any of the nucleic acids according to (i)    or (ii) or a complement thereof is a further embodiment of the    invention, and which preferably encodes a ACD protein that has    essentially the same biological activity as an ACD protein encoded    by SEQ ID NO: 2, 12, 14, 16, 18, 20, 22, 24, or 26; preferably the    encoded ACD protein confers enhanced fungal resistance relative to    control plants; and/or by-   (iv) an exogenous nucleic acid encoding the same ACD protein as the    ACD nucleic acids of (i) to (iii) above, but differing from the ACD    nucleic acids of (i) to (iii) above due to the degeneracy of the    genetic code.

Another preferred embodiment is a method for increasing fungalresistance, preferably resistance to Phacopsoracea, for example soy beanrust, in a plant, plant part, or plant cell, by increasing theexpression of a ACD protein or a functional fragment, orthologue,paralogue or homologue thereof wherein the ACD protein is encoded by

-   (i) an exogenous nucleic acid having at least 60% identity,    preferably at least 70% sequence identity, at least 80%, at least    90%, at least 95%, at least 98%, at least 99% sequence identity, or    even 100% sequence identity with SEQ ID NO: 1 or a functional    fragment thereof, an orthologue or a paralogue thereof;-   (ii) an exogenous nucleic acid encoding a protein having at least    60%, preferably at least 70% sequence identity, at least 80%, at    least 90%, at least 95%, at least 98%, at least 99% sequence    identity, or even 100% sequence identity with SEQ ID NO: 2, a    functional fragment thereof, an orthologue or a paralogue thereof;-   (iii) an exogenous nucleic acid capable of hybridizing under    stringent conditions with any of the nucleic acids according to (i)    or (ii) or a complement thereof is a further embodiment of the    invention, and which preferably encodes a ACD protein that has    essentially the same biological activity as an ACD protein encoded    by SEQ ID NO: 2; preferably the encoded ACD protein confers enhanced    fungal resistance relative to control plants; and/or by-   (iv) an exogenous nucleic acid encoding the same ACD protein as the    ACD nucleic acids of (i) to (iii) above, but differing from the ACD    nucleic acids of (i) to (iii) above due to the degeneracy of the    genetic code.

In a further method of the invention, the method comprises the steps of

-   (a) stably transforming a plant cell with a recombinant expression    cassette comprising    -   (i) a nucleic acid having at least 60% identity, preferably at        least 70% sequence identity, at least 80%, at least 90%, at        least 95%, at least 98%, at least 99% sequence identity, or even        100% sequence identity with SEQ ID NO: 1, 3-10, 11, 13, 15, 17,        19, 21, 23, or 25 or a functional fragment thereof, or an        orthologue or a paralogue thereof;    -   (ii) a nucleic acid coding for a protein having at least 60%        identity, preferably at least 70% sequence identity, at least        80%, at least 90%, at least 95%, at least 98%, at least 99%        sequence identity, or even 100% sequence identity with SEQ ID        NO: 2, 12, 14, 16, 18, 20, 22, 24, or 26, a functional fragment        thereof, an orthologue or a paralogue thereof;    -   (iii) a nucleic acid capable of hybridizing under stringent        conditions with any of the nucleic acids according to (i)        or (ii) or a complement thereof, and which preferably encodes a        ACD protein that has essentially the same biological activity as        an ACD protein encoded by SEQ ID NO: 2; preferably the encoded        ACD protein confers enhanced fungal resistance relative to        control plants; and/or by    -   (iv) a nucleic acid encoding the same ACD protein as the ACD        nucleic acids of (i) to (iii) above, but differing from the ACD        nucleic acids of (i) to (iii) above due to the degeneracy of the        genetic code;        in functional linkage with a promoter;-   (b) regenerating the plant from the plant cell; and-   (c) expressing said nucleic acid, optionally wherein the nucleic    acid which codes for a ACD protein is expressed in an amount and for    a period sufficient to generate or to increase soybean rust    resistance in said plant.

In a further method of the invention, the method comprises the steps of

-   (a) stably transforming a plant cell with a recombinant expression    cassette comprising    -   (i) a nucleic acid having at least 60% identity, preferably at        least 70% sequence identity, at least 80%, at least 90%, at        least 95%, at least 98%, at least 99% sequence identity, or even        100% sequence identity with SEQ ID NO: 1 or a functional        fragment thereof, or an orthologue or a paralogue thereof;    -   (ii) a nucleic acid coding for a protein having at least 60%        identity, preferably at least 70% sequence identity, at least        80%, at least 90%, at least 95%, at least 98%, at least 99%        sequence identity, or even 100% sequence identity with SEQ ID        NO: 2, a functional fragment thereof, an orthologue or a        paralogue thereof;    -   (iii) a nucleic acid capable of hybridizing under stringent        conditions with any of the nucleic acids according to (i)        or (ii) or a complement thereof, and which preferably encodes a        ACD protein that has essentially the same biological activity as        an ACD protein encoded by SEQ ID NO: 2; preferably the encoded        ACD protein confers enhanced fungal resistance relative to        control plants; and/or by    -   (iv) a nucleic acid encoding the same ACD protein as the ACD        nucleic acids of (i) to (iii) above, but differing from the ACD        nucleic acids of (i) to (iii) above due to the degeneracy of the        genetic code;        in functional linkage with a promoter;-   (b) regenerating the plant from the plant cell; and-   (c) expressing said nucleic acid, optionally wherein the nucleic    acid which codes for a ACD protein is expressed in an amount and for    a period sufficient to generate or to increase soybean rust    resistance in said plant.

A preferred embodiment is a method for increasing resistance to soy beanrust in a soy bean plant, soy bean plant part, or soy bean plant cell,by increasing the expression of a ACD protein, wherein the ACD proteinis encoded by

-   (i) an exogenous nucleic acid having at least 95%, at least 98%, at    least 99% sequence identity, or even 100% sequence identity with SEQ    ID NO: 1, 3-10, 11, 13, 15, 17, 19, 21, 23, or 25;-   (ii) an exogenous nucleic acid encoding a protein having at least    95%, at least 98%, at least 99% sequence identity, or even 100%    sequence identity with SEQ ID NO: 2, 12, 14, 16, 18, 20, 22, 24, or    26;-   (iii) an exogenous nucleic acid capable of hybridizing under    stringent conditions with any of the nucleic acids according to (i)    or (ii) or a complement thereof, and which preferably encodes a ACD    protein that has essentially the same biological activity as an ACD    protein encoded by SEQ ID NO: 2; preferably the encoded ACD protein    confers enhanced fungal resistance relative to control plants;    and/or by-   (iv) an exogenous nucleic acid encoding the same ACD protein as the    ACD nucleic acids of (i) to (iii) above, but differing from the ACD    nucleic acids of (i) to (iii) above due to the degeneracy of the    genetic code;    wherein increasing the expression of the ACD protein is achieved by    transforming the soy bean plant, plant part or plant cell with a    nucleic acid comprising the nucleic acid set out under item (i)    or (ii) or (iii).

A preferred embodiment is a method for increasing resistance to soy beanrust in a soy bean plant, soy bean plant part, or soy bean plant cell,by increasing the expression of a ACD protein, wherein the ACD proteinis encoded by

-   (i) an exogenous nucleic acid having at least 95%, at least 98%, at    least 99% sequence identity, or even 100% sequence identity with SEQ    ID NO: 1;-   (ii) an exogenous nucleic acid encoding a protein having at least    95%, at least 98%, at least 99% sequence identity, or even 100%    sequence identity with SEQ ID NO: 2;-   (iii) an exogenous nucleic acid capable of hybridizing under    stringent conditions with any of the nucleic acids according to (i)    or (ii) or a complement thereof, and which preferably encodes a ACD    protein that has essentially the same biological activity as an ACD    protein encoded by SEQ ID NO: 2; preferably the encoded ACD protein    confers enhanced fungal resistance relative to control plants;    and/or by-   (iv) an exogenous nucleic acid encoding the same ACD protein as the    ACD nucleic acids of (i) to (iii) above, but differing from the ACD    nucleic acids of (i) to (iii) above due to the degeneracy of the    genetic code;    wherein increasing the expression of the ACD protein is achieved by    transforming the soy bean plant, plant part or plant cell with a    nucleic acid comprising the nucleic acid set out under item (i)    or (ii) or (iii).

Also a preferred embodiment is a method for increasing resistance to soybean rust in a soy bean plant, soy bean plant part, or soy bean plantcell, by increasing the expression of a ACD protein, wherein the ACDprotein is encoded by

-   (i) an exogenous nucleic acid having at least 95%, at least 98%, at    least 99% sequence identity, or even 100% sequence identity with SEQ    ID NO: 1; or-   (ii) an exogenous nucleic acid encoding a protein having at least    95%, at least 98%, at least 99% sequence identity, or even 100%    sequence identity with SEQ ID NO: 2;    wherein increasing the expression of the ACD protein is achieved by    transforming the soy bean plant, plant part or plant cell with a    nucleic acid comprising the nucleic acid set out under item (i) or    (ii).

The fungal pathogens or fungus-like pathogens (such as, for example,Chromista) can belong to the group comprising Plasmodiophoramycota,Oomycota, Ascomycota, Chytridiomycetes, Zygomycetes, Basidiomycota orDeuteromycetes (Fungi imperfecti). Pathogens which may be mentioned byway of example, but not by limitation, are those detailed in Tables 2and 3, and the diseases which are associated with them.

TABLE 2 Diseases caused by biotrophic and/or heminecrotrophicphytopathogenic fungi Disease Pathogen Leaf rust Puccinia reconditaYellow rust P. striiformis Powdery mildew Erysiphe graminis/Blumeriagraminis Rust (common corn) Puccinia sorghi Rust (Southern corn)Puccinia polysora Tobacco leaf spot Cercospora nicotianae Rust (soybean)Phakopsora pachyrhizi, P. meibomiae Rust (tropical corn) Physopellapallescens, P. zeae = Angiopsora zeae

TABLE 3 Diseases caused by necrotrophic and/or hemibiotrophic fungi andOomycetes Disease Pathogen Plume blotch Septoria (Stagonospora) nodorumLeaf blotch Septoria tritici Ear fusarioses Fusarium spp. Late blightPhytophthora infestans Anthrocnose leaf blight Colletotrichumgraminicola (teleomorph: Anthracnose stalk rot Glomerella graminicolaPolitis); Glomerella tucumanensis (anamorph: Glomerella falcatum Went)Curvularia leaf spot Curvularia clavata, C. eragrostidis, = C. maculans(teleomorph: Cochliobolus eragrostidis), Curvularia inaequalis, C.intermedia (teleomorph: Cochliobolus intermedius), Curvularia lunata(teleomorph: Cochliobolus lunatus), Curvularia pallescens (teleomorph:Cochliobolus pallescens), Curvularia senegalensis, C. tuberculata(teleomorph: Cochliobolus tuberculatus) Didymella leaf spot Didymellaexitalis Diplodia leaf spot or streak Stenocarpella macrospora =Diplodialeaf macrospora Brown stripe downy Sclerophthora rayssiae var.zeae mildew Crazy top downy mildew Sclerophthora macrospora =Sclerospora macrospora Green ear downy mildew (graminicola Sclerosporagraminicola downy mildew) Leaf spots, minor Alternaria alternata,Ascochyta maydis, A. tritici, A. zeicola, Bipolaris victoriae =Helminthosporium victoriae (teleomorph: Cochliobolus victoriae), C.sativus (anamorph: Bipolaris sorokiniana = H. sorokinianum = H.sativum), Epicoccum nigrum, Exserohilum prolatum = Drechslera prolata(teleomorph: Setosphaeria prolata) Graphium penicillioides,Leptosphaeria maydis, Leptothyrium zeae, Ophiosphaerella herpotricha,(anamorph: Scolecosporiella sp.), Paraphaeosphaeria michotii, Phoma sp.,Septoria zeae, S. zeicola, S. zeina Northern corn leaf blight (whiteSetosphaeria turcica (anamorph: Exserohilum blast, crown stalk rot,stripe) turcicum = Helminthosporium turcicum) Northern corn leaf spotHelminthosporium Cochliobolus carbonum (anamorph: Bipolaris ear rot(race 1) zeicola = Helminthosporium carbonum) Phaeosphaeria leaf spotPhaeosphaeria maydis = Sphaerulina maydis Rostratum leaf spot(Helminthosporium Setosphaeria rostrata, (anamorph: leaf disease, earand xserohilum rostratum = Helminthosporium stalk rot) rostratum) Javadowny mildew Peronosclerospora maydis = Sclerospora maydis Philippinedowny mildew Peronosclerospora philippinensis = Sclerosporaphilippinensis Sorghum downy mildew Peronosclerospora sorghi =Sclerospora sorghi Spontaneum downy mildew Peronosclerospora spontanea =Sclerospora spontanea Sugarcane downy mildew Peronosclerospora sacchari= Sclerospora sacchari Sclerotium ear rot (southern blight) Sclerotiumrolfsii Sacc. (teleomorph: Athelia rolfsii) Seed rot-seedling blightBipolaris sorokiniana, B. zeicola = Helminthosporium carbonum, Diplodiamaydis, Exserohilum pedicillatum, Exserohilum turcicum =Helminthosporium turcicum, Fusarium avenaceum, F. culmorum, F.moniliforme, Gibberella zeae (anamorph: F. graminearum), Macrophominaphaseolina, Penicillium spp., Phomopsis sp., Pythium spp., Rhizoctoniasolani, R. zeae, Sclerotium rolfsii, Spicaria sp. Selenophoma leaf spotSelenophoma sp. Yellow leaf blight Ascochyta ischaemi, Phyllostictamaydis (teleomorph: Mycosphaerella zeae-maydis) Zonate leaf spotGloeocercospora sorghi

The following are especially preferred:

-   -   Plasmodiophoromycota such as Plasmodiophora brassicae (clubroot        of crucifers), Spongospora subterranea, Polymyxa graminis,    -   Oomycota such as Bremia lactucae (downy mildew of lettuce),        Peronospora (downy mildew) in snapdragon (P. antirrhini), onion        (P. destructor), spinach (P. effusa), soybean (P. manchurica),        tobacco (“blue mold”; P. tabacina) alfalfa and clover (P.        trifolium), Pseudoperonospora humuli (downy mildew of hops),        Plasmopara (downy mildew in grapevines) (P. viticola) and        sunflower (P. halstedii), Sclerophthora macrospora (downy mildew        in cereals and grasses), Pythium (for example damping-off of        Beta beet caused by P. debaryanum), Phytophthora infestans (late        blight in potato and in tomato and the like), Albugo spec.    -   Ascomycota such as Microdochium nivale (snow mold of rye and        wheat), Fusarium, Fusarium graminearum, Fusarium culmorum        (partial ear sterility mainly in wheat), Fusarium oxysporum        (Fusarium wilt of tomato), Blumeria graminis (powdery mildew of        barley (f.sp. hordei) and wheat (f.sp. tritici)), Erysiphe pisi        (powdery mildew of pea), Nectria galligena (Nectria canker of        fruit trees), Uncinula necator (powdery mildew of grapevine),        Pseudopeziza tracheiphila (red fire disease of grapevine),        Claviceps purpurea (ergot on, for example, rye and grasses),        Gaeumannomyces graminis (take-all on wheat, rye and other        grasses), Magnaporthe grisea, Pyrenophora graminea (leaf stripe        of barley), Pyrenophora teres (net blotch of barley),        Pyrenophora tritici-repentis (leaf blight of wheat), Venturia        inaequalis (apple scab), Sclerotinia sclerotium (stalk break,        stem rot), Pseudopeziza medicaginis (leaf spot of alfalfa, white        and red clover).    -   Basidiomycetes such as Typhula incarnata (typhula blight on        barley, rye, wheat), Ustilago maydis (blister smut on maize),        Ustilago nuda (loose smut on barley), Ustilago tritici (loose        smut on wheat, spelt), Ustilago avenae (loose smut on oats),        Rhizoctonia solani (rhizoctonia root rot of potato),        Sphacelotheca spp. (head smut of sorghum), Melampsora lini (rust        of flax), Puccinia graminis (stem rust of wheat, barley, rye,        oats), Puccinia recondita (leaf rust on wheat), Puccinia        dispersa (brown rust on rye), Puccinia hordei (leaf rust of        barley), Puccinia coronata (crown rust of oats), Puccinia        striiformis (yellow rust of wheat, barley, rye and a large        number of grasses), Uromyces appendiculatus (brown rust of        bean), Sclerotium rolfsii (root and stem rots of many plants).    -   Deuteromycetes (Fungi imperfecti) such as Septoria        (Stagonospora) nodorum (glume blotch) of wheat (Septoria        tritici), Pseudocercosporella herpotrichoides (eyespot of wheat,        barley, rye), Rynchosporium secalis (leaf spot on rye and        barley), Alternaria solani (early blight of potato, tomato),        Phoma betae (blackleg on Beta beet), Cercospora beticola (leaf        spot on Beta beet), Alternaria brassicae (black spot on oilseed        rape, cabbage and other crucifers), Verticillium dahliae        (verticillium wilt), Colletotrichum, Colletotrichum        lindemuthianum (bean anthracnose), Phoma lingam (blackleg of        cabbage and oilseed rape), Botrytis cinerea (grey mold of        grapevine, strawberry, tomato, hops and the like).

Especially preferred are biotrophic pathogens, e.g., Phakopsorapachyrhizi and/or those pathogens which have essentially a similarinfection mechanism as Phakopsora pachyrhizi, as described herein.Particularly preferred are pathogens from the subclass Pucciniomycetes,preferably from the order Pucciniales (rust), previously known asUredinales, among which in particular the Melompsoraceae. Preferred arePhakopsoraceae, more preferably Phakopsora. Especially preferred arePhakopsora pachyrhizi and/or Phakopsora meibomiae.

Also preferred rust fungi are selected from the group of Puccinia,Gymnosporangium, Juniperus, Cronartium, Hemlleia, and Uromyces,preferably Puccinia sorghi, Gymnosporangium juniperi-virginianae,Juniperus virginiana, Cronartium ribicola, Hemlleia vastatrix, Pucciniagraminis, Puccinia coronata, Uromyces phaseoli, Puccinia hemerocallidis,Puccinia persistens subsp. Triticina, Puccinia stniformis, Pucciniagraminis causes, and/or Uromyces appendeculatus.

A recombinant vector construct comprising:

-   (a) (i) a nucleic acid having at least 60% identity, preferably at    least 70% sequence identity, at least 80%, at least 90%, at least    95%, at least 98%, at least 99% sequence identity, or even 100%    sequence identity with SEQ ID NO: 1, 3-10, 11, 13, 15, 17, 19, 21,    23, or 25 or a functional fragment thereof, or an orthologue or a    paralogue thereof;    -   (ii) a nucleic acid coding for a protein having at least 60%        identity, preferably at least 70% sequence identity, at least        80%, at least 90%, at least 95%, at least 98%, at least 99%        sequence identity, or even 100% sequence identity with SEQ ID        NO: 2, 12, 14, 16, 18, 20, 22, 24, or 26, a functional fragment        thereof, an orthologue or a paralogue thereof;    -   (iii) a nucleic acid capable of hybridizing under stringent        conditions with any of the nucleic acids according to (i)        or (ii) or a complement thereof, and which preferably encodes a        ACD protein that has essentially the same biological activity as        an ACD protein encoded by SEQ ID NO: 2; preferably the encoded        ACD protein confers enhanced fungal resistance relative to        control plants; and/or by    -   (iv) a nucleic acid encoding the same ACD protein as the ACD        nucleic acids of (i) to (iii) above, but differing from the ACD        nucleic acids of (i) to (iii) above due to the degeneracy of the        genetic code        operably linked with-   (b) a promoter and-   (c) a transcription termination sequence is a further embodiment of    the invention.

A recombinant vector construct comprising:

-   (a) (i) a nucleic acid having at least 60% identity, preferably at    least 70% sequence identity, at least 80%, at least 90%, at least    95%, at least 98%, at least 99% sequence identity, or even 100%    sequence identity with SEQ ID NO: 1 or a functional fragment    thereof, or an orthologue or a paralogue thereof;    -   (ii) a nucleic acid coding for a protein having at least 60%        identity, preferably at least 70% sequence identity, at least        80%, at least 90%, at least 95%, at least 98%, at least 99%        sequence identity, or even 100% sequence identity with SEQ ID        NO: 2, a functional fragment thereof, an orthologue or a        paralogue thereof;    -   (iii) a nucleic acid capable of hybridizing under stringent        conditions with any of the nucleic acids according to (i)        or (ii) or a complement thereof, and which preferably encodes a        ACD protein that has essentially the same biological activity as        an ACD protein encoded by SEQ ID NO: 2; preferably the encoded        ACD protein confers enhanced fungal resistance relative to        control plants; and/or by    -   (iv) a nucleic acid encoding the same ACD protein as the ACD        nucleic acids of (i) to (iii) above, but differing from the ACD        nucleic acids of (i) to (iii) above due to the degeneracy of the        genetic code        operably linked with-   (b) a promoter and-   (c) a transcription termination sequence is a further embodiment of    the invention.

Furthermore, a recombinant vector construct is provided comprising:

-   (a) (i) a nucleic acid having at least 95%, at least 98%, at least    99% sequence identity, or even 100% sequence identity with SEQ ID    NO: 1, 3-10, 11, 13, 15, 17, 19, 21, 23, or 25;    -   (ii) a nucleic acid coding for a protein having at least 95%, at        least 98%, at least 99% sequence identity, or even 100% sequence        identity with SEQ ID NO: 2, 12, 14, 16, 18, 20, 22, 24, or 26;    -   (iii) a nucleic acid capable of hybridizing under stringent        conditions with any of the nucleic acids according to (i)        or (ii) or a complement thereof, and which preferably encodes a        ACD protein that has essentially the same biological activity as        an ACD protein encoded by SEQ ID NO: 2; preferably the encoded        ACD protein confers enhanced fungal resistance relative to        control plants; and/or by    -   (iv) a nucleic acid encoding the same ACD protein as the ACD        nucleic acids of (i) to (iii) above, but differing from the ACD        nucleic acids of (i) to (iii) above due to the degeneracy of the        genetic code        operably linked with-   (b) a promoter and-   (c) a transcription termination sequence is a further embodiment of    the invention.

Furthermore, a recombinant vector construct is provided comprising:

-   (a) (i) a nucleic acid having at least 95%, at least 98%, at least    99% sequence identity, or even 100% sequence identity with SEQ ID    NO: 1;    -   (ii) a nucleic acid coding for a protein having at least 95%, at        least 98%, at least 99% sequence identity, or even 100% sequence        identity with SEQ ID NO: 2;    -   (iii) a nucleic acid capable of hybridizing under stringent        conditions with any of the nucleic acids according to (i)        or (ii) or a complement thereof, and which preferably encodes a        ACD protein that has essentially the same biological activity as        an ACD protein encoded by SEQ ID NO: 2; preferably the encoded        ACD protein confers enhanced fungal resistance relative to        control plants; and/or by    -   (iv) a nucleic acid encoding the same ACD protein as the ACD        nucleic acids of (i) to (iii) above, but differing from the ACD        nucleic acids of (i) to (iii) above due to the degeneracy of the        genetic code        operably linked with-   (b) a promoter and-   (c) a transcription termination sequence is a further embodiment of    the invention.

Promoters according to the present invention may be constitutive,inducible, in particular pathogen-inducible, developmentalstage-preferred, cell type-preferred, tissue-preferred ororgan-preferred. Constitutive promoters are active under mostconditions. Non-limiting examples of constitutive promoters include theCaMV 19S and 35S promoters (Odell et al., 1985, Nature 313:810-812), thesX CaMV 35S promoter (Kay et al., 1987, Science 236:1299-1302), the Sep1promoter, the rice actin promoter (McElroy et al., 1990, Plant Cell2:163-171), the Arabidopsis actin promoter, the ubiquitin promoter(Christensen et al., 1989, Plant Molec. Biol. 18:675-689); pEmu (Last etal., 1991, Theor. Appl. Genet. 81:581-588), the figwort mosaic virus 35Spromoter, the Smas promoter (Velten et al., 1984, EMBO J. 3:2723-2730),the GRP1-8 promoter, the cinnamyl alcohol dehydrogenase promoter (U.S.Pat. No. 5,683,439), promoters from the T-DNA of Agrobacterium, such asmannopine synthase, nopaline synthase, and octopine synthase, the smallsubunit of ribulose biphosphate carboxylase (ssuRUBISCO) promoter,and/or the like.

Preferably, the expression vector of the invention comprises aconstitutive promoter, mesophyll-specific promoter, epidermis-specificpromoter, root-specific promoter, a pathogen inducible promoter, or afungal-inducible promoter. A promoter is inducible, if its activity,measured on the amount of RNA produced under control of the promoter, isat least 30%, at least 40%, at least 50% preferably at least 60%, atleast 70%, at least 80%, at least 90% more preferred at least 100%, atleast 200%, at least 300% higher in its induced state, than in itsun-induced state. A promoter is cell-, tissue- or organ-specific, if itsactivity, measured on the amount of RNA produced under control of thepromoter, is at least 30%, at least 40%, at least 50% preferably atleast 60%, at least 70%, at least 80%, at least 90% more preferred atleast 100%, at least 200%, at least 300% higher in a particularcell-type, tissue or organ, then in other cell-types or tissues of thesame plant, preferably the other cell-types or tissues are cell types ortissues of the same plant organ, e.g. a root. In the case of organspecific promoters, the promoter activity has to be compared to thepromoter activity in other plant organs, e.g. leaves, stems, flowers orseeds. Preferably, the promoter is a constitutive promoter,mesophyll-specific promoter, or epidermis-specific promoter.

In preferred embodiments, the increase in the protein amount and/oractivity of the ACD protein takes place in a constitutive ortissue-specific manner. In especially preferred embodiments, anessentially pathogen-induced increase in the protein amount and/orprotein activity takes place, for example by recombinant expression ofthe ACD nucleic acid under the control of a fungal-inducable promoter.In particular, the expression of the ACD nucleic acid takes place onfungal infected sites, where, however, preferably the expression of theACD nucleic acid remains essentially unchanged in tissues not infectedby fungus.

Developmental stage-preferred promoters are preferentially expressed atcertain stages of development. Tissue and organ preferred promotersinclude those that are preferentially expressed in certain tissues ororgans, such as leaves, roots, seeds, or xylem. Examples of tissuepreferred and organ preferred promoters include, but are not limited tofruit-preferred, ovule-preferred, male tissue-preferred, seed-preferred,integument-preferred, tuber-preferred, stalk-preferred,pericarp-preferred, leaf-preferred, stigma-preferred, pollen-preferred,anther-preferred, a petal-preferred, sepal-preferred, pedicel-preferred,silique-preferred, stem-preferred, root-preferred promoters and/or thelike. Seed preferred promoters are preferentially expressed during seeddevelopment and/or germination. For example, seed preferred promoterscan be embryo-preferred, endosperm preferred and seed coat-preferred.See Thompson et al., 1989, BioEssays 10:108. Examples of seed preferredpromoters include, but are not limited to cellulose synthase (celA),Cim1, gamma-zein, globulin-1, maize 19 kD zein (cZ19B1) and/or the like.

Other suitable tissue-preferred or organ-preferred promoters include,but are not limited to, the napin-gene promoter from rapeseed (U.S. Pat.No. 5,608,152), the USP-promoter from Vicia faba (Baeumlein et al.,1991, Mol Gen Genet. 225(3):459-67), the oleosin-promoter fromArabidopsis (PCT Application No. WO 98/45461), the phaseolin-promoterfrom Phaseolus vulgaris (U.S. Pat. No. 5,504,200), the Bce4-promoterfrom Brassica (PCT Application No. WO 91/13980), or the legumin B4promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2(2):233-9), aswell as promoters conferring seed specific expression in monocot plantslike maize, barley, wheat, rye, rice, etc. Suitable promoters to noteare the Ipt2 or Ipt1-gene promoter from barley (PCT Application No. WO95/15389 and PCT Application No. WO 95/23230) or those described in PCTApplication No. WO 99/16890 (promoters from the barley hordein-gene,rice glutelin gene, rice oryzin gene, rice prolamin gene, wheat gliadingene, wheat glutelin gene, oat glutelin gene, Sorghum kasirin-gene,and/or rye secalin gene)

Promoters useful according to the invention include, but are not limitedto, are the major chlorophyll a/b binding protein promoter, histonepromoters, the Ap3 promoter, the β-conglycin promoter, the napinpromoter, the soybean lectin promoter, the maize 15 kD zein promoter,the 22 kD zein promoter, the 27 kD zein promoter, the g-zein promoter,the waxy, shrunken 1, shrunken 2, bronze promoters, the Zm13 promoter(U.S. Pat. No. 5,086,169), the maize polygalacturonase promoters (PG)(U.S. Pat. Nos. 5,412,085 and 5,545,546), the SGB6 promoter (U.S. Pat.No. 5,470,359), as well as synthetic or other natural promoters.

Epidermis-specific promoters may be selected from the group consistingof:

WIR5 (=GstA1); acc. X56012; Dudler & Schweizer,

GLP4, acc. AJ310534; Wei Y., Zhang Z., Andersen C. H., Schmelzer E.,Gregersen P. L., Collinge D. B., Smedegaard-Petersen V. andThordal-Christensen H., Plant Molecular Biology 36, 101 (1998),

GLP2a, acc. AJ237942, Schweizer P., Christoffel A. and Dudler R., PlantJ. 20, 541 (1999);

Prx7, acc. AJ003141, Kristensen B. K., Ammitzböll H., Rasmussen S. K.and Nielsen K. A., Molecular Plant Pathology, 2(6), 311 (2001);

GerA, acc. AF250933; Wu S., Druka A., Horvath H., Kleinhofs A.,Kannangara G. and von Wettstein D., Plant Phys Biochem 38, 685 (2000);

OsROC1, acc. AP004656

RTBV, acc. AAV62708, AAV62707; Klöti A., Henrich C., Bieri S., He X.,Chen G., Burkhardt P. K., Minn J., Lucca P., Hohn T., Potrykus I. andFütterer J., PMB 40, 249 (1999);

Chitinase ChtC2-Promoter from potato (Ancillo et al., Planta. 217(4),566, (2003));

AtProT3 Promoter (Grallath et al., Plant Physiology. 137(1), 117(2005));

SHN-Promoters from Arabidopsis (AP2/EREBP transcription factors involvedin cutin and wax production) (Aarón et al., Plant Cell. 16(9), 2463(2004)); and/or

GSTA1 from wheat (Dudler et al., WP2005306368 and Altpeter et al., PlantMolecular Biology. 57(2), 271 (2005)).

Mesophyll-specific promoters may be selected from the group consistingof:

PPCZm1 (=PEPC); Kausch A. P., Owen T. P., Zachwieja S. J., Flynn A. R.and Sheen J., Plant Mol. Biol. 45, 1 (2001);

OsrbcS, Kyozuka et al., PlaNT Phys 102, 991 (1993); Kyozuka J., McElroyD., Hayakawa T., Xie Y., Wu R. and Shimamoto K., Plant Phys. 102, 991(1993);

OsPPDK, acc. AC099041;

TaGF-2.8, acc. M63223; Schweizer P., Christoffel A. and Dudler R., PlantJ. 20, 541 (1999);

TaFBPase, acc. X53957;

TaWIS1, acc. AF467542; US 200220115849;

HvBIS1, acc. AF467539; US 200220115849;

ZmMIS1, acc. AF467514; US 200220115849;

HvPR1a, acc. X74939; Bryngelsson et al., Mol. Plant Microbe Interacti. 7(2), 267 (1994);

HvPR1b, acc. X74940; Bryngelsson et al., Mol. Plant Microbe Interact.7(2), 267 (1994);

HvB1,3gluc; acc. AF479647;

HvPrx8, acc. AJ276227; Kristensen et al., Molecular Plant Pathology,2(6), 311 (2001); and/or

HvPAL, acc. X97313; Wei Y., Zhang Z., Andersen C. H., Schmelzer E.,Gregersen P. L., Collinge D. B., Smedegaard-Petersen V. andThordal-Christensen H. Plant Molecular Biology 36, 101 (1998).

Constitutive promoters may be selected from the group consisting of

-   -   PcUbi promoter from parsley (WO 03/102198)    -   CaMV 35S promoter: Cauliflower Mosaic Virus 35S promoter (Benfey        et al. 1989 EMBO J. 8(8): 2195-2202),    -   STPT promoter: Arabidopsis thaliana Short Triose phosphat        translocator promoter (Accession NM_123979)    -   Act1 promoter:—Oryza sativa actin 1 gene promoter (McElroy et        al. 1990 PLANT CELL 2(2) 163-171 a) and/or    -   EF1A2 promoter: Glycine max translation elongation factor EF1        alpha (US 20090133159).

One type of vector construct is a “plasmid,” which refers to a circulardouble stranded DNA loop into which additional DNA segments can beligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectorconstructs are capable of autonomous replication in a host plant cellinto which they are introduced. Other vector constructs are integratedinto the genome of a host plant cell upon introduction into the hostcell, and thereby are replicated along with the host genome. Inparticular the vector construct is capable of directing the expressionof gene to which the vectors is operatively linked. However, theinvention is intended to include such other forms of expression vectorconstructs, such as viral vectors (e.g., potato virus X, tobacco rattlevirus, and/or Gemini virus), which serve equivalent functions.

In preferred embodiments, the increase in the protein quantity orfunction of the ACD protein takes place in a constitutive ortissue-specific manner. In especially preferred embodiments, anessentially pathogen-induced increase in the protein quantity or proteinfunction takes place, for example by exogenous expression of the ACDnucleic acid under the control of a fungal-inducible promoter. Inparticular, the expression of the ACD nucleic acid takes place on fungalinfected sites, where, however, preferably the expression of the ACDnucleic acid sequence remains essentially unchanged in tissues notinfected by fungus. In preferred embodiments, the protein amount of aACD protein in the plant is increased by at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or at least 95% or more in comparison to a wildtype plant that is not transformed with the ACD nucleic acid.

A preferred embodiment is a transgenic plant, transgenic plant part, ortransgenic plant cell overexpressing an exogenous ACD protein.Preferably, the ACD protein overexpressed in the plant, plant part orplant cell is encoded by

-   (i) an exogenous nucleic acid having at least 60% identity with SEQ    ID NO: 1, 3-10, 11, 13, 15, 17, 19, 21, 23, or 25 or a functional    fragment, thereof, an orthologue or a paralogue thereof; or by-   (ii) an exogenous nucleic acid encoding a protein having at least    60% identity with SEQ ID NO: 2, 12, 14, 16, 18, 20, 22, 24, or 26, a    functional fragment thereof, an orthologue or a paralogue thereof;-   (iii) an exogenous nucleic acid capable of hybridizing under    stringent conditions with any of the nucleic acids according to (i)    or (ii) or a complement thereof, and which preferably encodes a ACD    protein that has essentially the same biological activity as an ACD    protein encoded by SEQ ID NO: 2; preferably the encoded ACD protein    confers enhanced fungal resistance relative to control plants. Most    preferably, the exogenous nucleic acid has at least 95%, at least    98%, at least 99% sequence identity, or even 100% sequence identity    with SEQ ID NO: 1; or comprises an exogenous nucleic acid encoding a    protein having at least 95%, at least 98%, at least 99% sequence    identity, or even 100% sequence identity with SEQ ID NO: 2; and/or    by-   (iv) an exogenous nucleic acid encoding the same ACD protein as the    ACD nucleic acids of (i) to (iii) above, but differing from the ACD    nucleic acids of (i) to (iii) above due to the degeneracy of the    genetic code.

A preferred embodiment is a transgenic plant, transgenic plant part, ortransgenic plant cell overexpressing an exogenous ACD protein.Preferably, the ACD protein overexpressed in the plant, plant part orplant cell is encoded by

-   (i) an exogenous nucleic acid having at least 60% identity with SEQ    ID NO: 1 or a functional fragment, thereof, an orthologue or a    paralogue thereof; or by-   (ii) an exogenous nucleic acid encoding a protein having at least    60% identity with SEQ ID NO: 2, a functional fragment thereof, an    orthologue or a paralogue thereof;-   (iii) an exogenous nucleic acid capable of hybridizing under    stringent conditions with any of the nucleic acids according to (i)    or (ii) or a complement thereof, and which preferably encodes a ACD    protein that has essentially the same biological activity as an ACD    protein encoded by SEQ ID NO: 2; preferably the encoded ACD protein    confers enhanced fungal resistance relative to control plants. Most    preferably, the exogenous nucleic acid has at least 95%, at least    98%, at least 99% sequence identity, or even 100% sequence identity    with SEQ ID NO: 1; or comprises an exogenous nucleic acid encoding a    protein having at least 95%, at least 98%, at least 99% sequence    identity, or even 100% sequence identity with SEQ ID NO: 2; and/or    by-   (iv) a nucleic acid encoding the same ACD protein as the ACD nucleic    acids of (i) to (iii) above, but differing from the ACD nucleic    acids of (i) to (iii) above due to the degeneracy of the genetic    code.

More preferably, the transgenic plant, transgenic plant part, ortransgenic plant cell according to the present invention has beenobtained by transformation with a recombinant vector described herein.

Suitable methods for transforming or transfecting host cells includingplant cells are well known in the art of plant biotechnology. Any methodmay be used to transform the recombinant expression vector into plantcells to yield the transgenic plants of the invention. General methodsfor transforming dicotyledonous plants are disclosed, for example, inU.S. Pat. Nos. 4,940,838; 5,464,763, and the like. Methods fortransforming specific dicotyledonous plants, for example, cotton, areset forth in U.S. Pat. Nos. 5,004,863; 5,159,135; and 5,846,797. Soytransformation methods are set forth in U.S. Pat. Nos. 4,992,375;5,416,011; 5,569,834; 5,824,877; 6,384,301 and in EP 0301749B1 may beused. Transformation methods may include direct and indirect methods oftransformation. Suitable direct methods include polyethylene glycolinduced DNA uptake, liposome-mediated transformation (U.S. Pat. No.4,536,475), biolistic methods using the gene gun (Fromm M E et al.,Bio/Technology. 8(9):833-9, 1990; Gordon-Kamm et al. Plant Cell 2:603,1990), electroporation, incubation of dry embryos in DNA-comprisingsolution, and microinjection. In the case of these direct transformationmethods, the plasmids used need not meet any particular requirements.Simple plasmids, such as those of the pUC series, pBR322, M13mp series,pACYC184 and the like can be used. If intact plants are to beregenerated from the transformed cells, an additional selectable markergene is preferably located on the plasmid. The direct transformationtechniques are equally suitable for dicotyledonous and monocotyledonousplants.

Transformation can also be carried out by bacterial infection by meansof Agrobacterium (for example EP 0 116 718), viral infection by means ofviral vectors (EP 0 067 553; U.S. Pat. No. 4,407,956; WO 95/34668; WO93/03161) or by means of pollen (EP 0 270 356; WO 85/01856; U.S. Pat.No. 4,684,611). Agrobacterium based transformation techniques(especially for dicotyledonous plants) are well known in the art. TheAgrobacterium strain (e.g., Agrobacterium tumefaciens or Agrobacteriumrhizogenes) comprises a plasmid (Ti or Ri plasmid) and a T-DNA elementwhich is transferred to the plant following infection withAgrobacterium.

The T-DNA (transferred DNA) is integrated into the genome of the plantcell. The T-DNA may be localized on the Ri- or Ti-plasmid or isseparately comprised in a so-called binary vector. Methods for theAgrobacterium-mediated transformation are described, for example, inHorsch R B et al. (1985) Science 225:1229. The Agrobacterium-mediatedtransformation is best suited to dicotyledonous plants but has also beenadapted to monocotyledonous plants. The transformation of plants byAgrobacteria is described in, for example, White F F, Vectors for GeneTransfer in Higher Plants, Transgenic Plants, Vol. 1, Engineering andUtilization, edited by S. D. Kung and R. Wu, Academic Press, 1993, pp.15-38; Jenes B et al. Techniques for Gene Transfer, Transgenic Plants,Vol. 1, Engineering and Utilization, edited by S. D. Kung and R. Wu,Academic Press, 1993, pp. 128-143; Potrykus (1991) Annu Rev PlantPhysiol Plant Molec Biol 42:205-225. Transformation may result intransient or stable transformation and expression. Although a nucleotidesequence of the present invention can be inserted into any plant andplant cell falling within these broad classes, it is particularly usefulin crop plant cells.

The genetically modified plant cells can be regenerated via all methodswith which the skilled worker is familiar. Suitable methods can be foundin the abovementioned publications by S. D. Kung and R. Wu, Potrykus orHofgen and Willmitzer.

After transformation, plant cells or cell groupings may be selected forthe presence of one or more markers which are encoded byplant-expressible genes co-transferred with the gene of interest,following which the transformed material is regenerated into a wholeplant. To select transformed plants, the plant material obtained in thetransformation is, as a rule, subjected to selective conditions so thattransformed plants can be distinguished from untransformed plants. Forexample, the seeds obtained in the above-described manner can be plantedand, after an initial growing period, subjected to a suitable selectionby spraying. A further possibility consists in growing the seeds, ifappropriate after sterilization, on agar plates using a suitableselection agent so that only the transformed seeds can grow into plants.Alternatively, the transformed plants are screened for the presence of aselectable marker such as the ones described above. The transformedplants may also be directly selected by screening for the presence ofthe ACD nucleic acid.

Following DNA transfer and regeneration, putatively transformed plantsmay also be evaluated, for instance using Southern analysis, for thepresence of the gene of interest, copy number and/or genomicorganisation. Alternatively or additionally, expression levels of thenewly introduced DNA may be monitored using Northern and/or Westernanalysis, both techniques being well known to persons having ordinaryskill in the art.

The generated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plant may be selfedand homozygous second-generation (or T2) transformants selected, and theT2 plants may then further be propagated through classical breedingtechniques. The generated transformed organisms may take a variety offorms. For example, they may be chimeras of transformed cells andnon-transformed cells; clonal transformants (e.g., all cells transformedto contain the expression cassette); grafts of transformed anduntransformed tissues (e.g., in plants, a transformed rootstock graftedto an untransformed scion).

Preferably, the transgenic plant of the present invention or the plantobtained by the method of the present invention has increased resistanceagainst fungal pathogens, preferably against fungal pathogens of thefamily Phacopsoraceae, more preferably against fungal pathogens of thegenus Phacopsora, most preferably against Phakopsora pachyrhizi (Sydow)and Phakopsora meibomiae (Arthur), also known as soybean rust.Preferably, resistance against Phakopsora pachyrhizi (Sydow) and/orPhakopsora meibomiae (Arthur) is increased.

Preferably, the plant, plant part, or plant cell is a plant or derivedfrom a plant selected from the group consisting of beans, soya, pea,clover, kudzu, lucerne, lentils, lupins, vetches, groundnut, rice,wheat, barley, arabidopsis, lentil, banana, canola, cotton, potatoe,corn, sugar cane, alfalfa, and sugar beet.

In one embodiment of the present invention the plant is selected fromthe group consisting of beans, soya, pea, clover, kudzu, lucerne,lentils, lupins, vetches, and/or groundnut. Preferably, the plant is alegume, comprising plants of the genus Phaseolus (comprising Frenchbean, dwarf bean, climbing bean (Phaseolus vulgaris), Lima bean(Phaseolus lunatus L.), Tepary bean (Phaseolus acutifolius A. Gray),runner bean (Phaseolus coccineus)); the genus Glycine (comprisingGlycine soja, soybeans (Glycine max (L.) Merill)); pea (Pisum)(comprising shelling peas (Pisum sativum L. convar. sativum), alsocalled smooth or round-seeded peas; marrowfat pea (Pisum sativum L.convar. medullare Alef. emend. C. O. Lehm), sugar pea (Pisum sativum L.convar. axiphium Alef emend. C. O. Lehm), also called snow pea,edible-podded pea or mangetout, (Pisum granda sneida L. convar. sneidulop. shneiderium)); peanut (Arachis hypogaea), clover (Trifolium spec.),medick (Medicago), kudzu vine (Pueraria lobata), common lucerne, alfalfa(M. sativa L.), chickpea (Cicer), lentils (Lens) (Lens culinarisMedik.), lupins (Lupinus); vetches (Vicia), field bean, broad bean(Vicia faba), vetchling (Lathyrus) (comprising chickling pea (Lathyrussativus), heath pea (Lathyrus tuberosus)); genus Vigna (comprising mothbean (Vigna aconitifolia (Jacq.) Maréchal), adzuki bean (Vigna angularis(Willd.) Ohwi & H. Ohashi), urd bean (Vigna mungo (L.) Hepper), mungbean (Vigna radiata (L.) R. Wilczek), bambara groundnut (Vignasubterrane (L.) Verdc.), rice bean (Vigna umbellata (Thunb.) Ohwi & H.Ohashi), Vigna vexillata (L.) A. Rich., Vigna unguiculata (L.) Walp., inthe three subspecies asparagus bean, cowpea, catjang bean)); pigeonpea(Cajanus cajan (L.) Millsp.), the genus Macrotyloma (comprising geocarpagroundnut (Macrotyloma geocarpum (Harms) Maréchal & Baudet), horse bean(Macrotyloma uniflorum (Lam.) Verdc.)); goa bean (Psophocarpustetragonolobus (L.) DC.), African yam bean (Sphenostylis stenocarpa(Hochst. ex A. Rich.) Harms), Egyptian black bean, dolichos bean, lablabbean (Lablab purpureus (L.) Sweet), yam bean (Pachyrhizus), guar bean(Cyamopsis tetragonolobus (L.) Taub.); and/or the genus Canavalia(comprising jack bean (Canavalia ensiformis (L.) DC.), sword bean(Canavalia gladiata (Jacq.) DC.)).

Further preferred is a plant selected from plant is selected from thegroup consisting of beans, soya, pea, clover, kudzu, lucerne, lentils,lupins, vetches, and groundnut. Most preferably, the plant, plant part,or plant cell is or is derived from soy.

One embodiment according to the present invention provides a method forproducing a transgenic plant, a transgenic plant part, or a transgenicplant cell resistant to a fungal pathogen, preferably of the familyPhacosporaceae, for example soybean rust, wherein the recombinantnucleic acid used to generate a transgenic plant comprises a promoterthat is functional in the plant cell, operably linked to a ACD nucleicacid, which is preferably SEQ ID NO: 1, and

a terminator regulatory sequence.

In one embodiment, the present invention refers to a method for theproduction of a transgenic plant, transgenic plant part, or transgenicplant cell having increased fungal resistance, comprising

-   (a) introducing a recombinant vector construct according to the    present invention into a plant, a plant part or a plant cell and-   (b) generating a transgenic plant from the plant, plant part or    plant cell.

Preferably, the method for the production of the transgenic plant,transgenic plant part, or transgenic plant cell further comprises thestep

-   (c) expressing the ACD protein, preferably encoded by    -   (i) an exogenous nucleic acid having at least 60% identity with        SEQ ID NO: 1, 3-10, 11, 13, 15, 17, 19, 21, 23, or 25, a        functional fragment thereof, an orthologue or a paralogue        thereof;    -   (ii) an exogenous nucleic acid encoding a protein having at        least 60% identity with SEQ ID NO: 2, 12, 14, 16, 18, 20, 22,        24, or 26, or a functional fragment thereof, an orthologue or a        paralogue thereof;    -   (iii) an exogenous nucleic acid capable of hybridizing under        stringent conditions with any of the nucleic acids according        to (i) or (ii) or a complement thereof, and which preferably        encodes a ACD protein that has essentially the same biological        activity as an ACD protein encoded by SEQ ID NO: 2; preferably        the encoded ACD protein confers enhanced fungal resistance        relative to control plants; and/or by    -   (iv) an exogenous nucleic acid encoding the same ACD protein as        the ACD nucleic acids of (i) to (iii) above, but differing from        the ACD nucleic acids of (i) to (iii) above due to the        degeneracy of the genetic code.

Preferably, the method for the production of the transgenic plant,transgenic plant part, or transgenic plant cell further comprises thestep

-   (c) expressing the ACD protein, preferably encoded by    -   (i) an exogenous nucleic acid having at least 60% identity with        SEQ ID NO: 1, a functional fragment thereof, an orthologue or a        paralogue thereof;    -   (ii) an exogenous nucleic acid encoding a protein having at        least 60% identity with SEQ ID NO: 2, or a functional fragment        thereof, an orthologue or a paralogue thereof;    -   (iii) an exogenous nucleic acid capable of hybridizing under        stringent conditions with any of the nucleic acids according        to (i) or (ii) or a complement thereof, and which preferably        encodes a ACD protein that has essentially the same biological        activity as an ACD protein encoded by SEQ ID NO: 2; preferably        the encoded ACD protein confers enhanced fungal resistance        relative to control plants; and/or by    -   (iv) an exogenous nucleic acid encoding the same ACD protein as        the ACD nucleic acids of (i) to (iii) above, but differing from        the ACD nucleic acids of (i) to (iii) above due to the        degeneracy of the genetic code.

Preferably, the method for the production of the transgenic plant,transgenic plant part, or transgenic plant cell additionally comprisesthe step of harvesting the seeds of the transgenic plant and plantingthe seeds and growing the seeds to plants, wherein the grown plant(s)comprises

-   -   (i) the exogenous nucleic acid having at least 60% identity with        SEQ ID NO: 1, 3-10, 11, 13, 15, 17, 19, 21, 23, or 25, a        functional fragment thereof, an orthologue or a paralogue        thereof;    -   (ii) the exogenous nucleic acid encoding a protein having at        least 60% identity with SEQ ID NO: 2, 12, 14, 16, 18, 20, 22,        24, or 26, or a functional fragment thereof, an orthologue or a        paralogue thereof;    -   (iii) the exogenous nucleic acid capable of hybridizing under        stringent conditions with any of the nucleic acids according        to (i) or (ii) or a complement thereof, and which preferably        encodes a ACD protein that has essentially the same biological        activity as an ACD protein encoded by SEQ ID NO: 2; preferably        the encoded ACD protein confers enhanced fungal resistance        relative to control plants; and/or by    -   (iv) the exogenous nucleic acid encoding the same ACD protein as        the ACD nucleic acids of (i) to (iii) above, but differing from        the ACD nucleic acids of (i) to (iii) above due to the        degeneracy of the genetic code.

Preferably, the method for the production of the transgenic plant,transgenic plant part, or transgenic plant cell additionally comprisesthe step of harvesting the seeds of the transgenic plant and plantingthe seeds and growing the seeds to plants, wherein the grown plant(s)comprises

-   -   (i) the exogenous nucleic acid having at least 60% identity with        SEQ ID NO: 1, a functional fragment thereof, an orthologue or a        paralogue thereof;    -   (ii) the exogenous nucleic acid encoding a protein having at        least 60% identity with SEQ ID NO: 2, or a functional fragment        thereof, an orthologue or a paralogue thereof;    -   (iii) the exogenous nucleic acid capable of hybridizing under        stringent conditions with any of the nucleic acids according        to (i) or (ii) or a complement thereof, and which preferably        encodes a ACD protein that has essentially the same biological        activity as an ACD protein encoded by SEQ ID NO: 2; preferably        the encoded ACD protein confers enhanced fungal resistance        relative to control plants; and/or by    -   (iv) the exogenous nucleic acid encoding the same ACD protein as        the ACD nucleic acids of (i) to (iii) above, but differing from        the ACD nucleic acids of (i) to (iii) above due to the        degeneracy of the genetic code.

The transgenic plants may be selected by known methods as describedabove (e.g., by screening for the presence of one or more markers whichare encoded by plant-expressible genes co-transferred with the ACD geneor by directly screening for the ACD nucleic acid).

Furthermore, the use of the exogenous ACD nucleic acid or therecombinant vector construct comprising the ACD nucleic acid for thetransformation of a plant, plant part, or plant cell to provide a fungalresistant plant, plant part, or plant cell is provided.

Harvestable parts of the transgenic plant according to the presentinvention are part of the invention. The harvestable parts may be seeds,roots, leaves and/or flowers comprising the ACD nucleic acid or ACDprotein or parts thereof. Preferred parts of soy plants are soy beanscomprising the ACD nucleic acid or ACD protein.

Products derived from a transgenic plant according to the presentinvention, parts thereof or harvestable parts thereof are part of theinvention. A preferred product is soybean meal or soybean oil.

Preferably, the harvestable part of the transgenic plant or the productderived from the transgenic plant comprises an exogenous ACD nucleicacid, wherein the exogenous ACD nucleic acid is selected from the groupconsisting of:

-   -   (i) an exogenous nucleic acid having at least 60%, preferably at        least 70%, for example at least 75%, more preferably at least        80%, for example at least 85%, even more preferably at least        90%, for example at least 95% or at least 96% or at least 97% or        at least 98% most preferably 99% identity with SEQ ID NO: 1,        3-10, 11, 13, 15, 17, 19, 21, 23, or 25, a functional fragment        thereof, or an orthologue or a paralogue thereof; or by    -   (ii) an exogenous nucleic acid encoding a protein having at        least 60% identity, preferably at least 70%, for example at        least 75%, more preferably at least 80%, for example at least        85%, even more preferably at least 90%, for example at least 95%        or at least 96% or at least 97% or at least 98% most preferably        99% homology with SEQ ID NO: 2, 12, 14, 16, 18, 20, 22, 24, or        26, a functional fragment thereof, an orthologue or a paralogue        thereof,    -   (iii) an exogenous nucleic acid capable of hybridizing under        stringent conditions with any of the nucleic acids according        to (i) or (ii) or a complementary sequence (complement) thereof,        and which preferably encodes a ACD protein that has essentially        the same biological activity as an ACD protein encoded by SEQ ID        NO: 2; preferably the encoded ACD protein confers enhanced        fungal resistance relative to control plants; or by    -   (iv) an exogenous nucleic acid encoding the same ACD protein as        the ACD nucleic acids of (i) to (iii) above, but differing from        the ACD nucleic acids of (i) to (iii) above due to the        degeneracy of the genetic code;        or wherein the harvestable part of the transgenic plant or the        product derived from the transgenic plant comprises an ACD        protein encoded by any one of the ACD nucleic acids of (i) to        (iv).

In one embodiment the method for the production of a product comprises

-   a) growing the plants of the invention or obtainable by the methods    of invention and-   b) producing said product from or by the plants of the invention    and/or parts, e.g. seeds, of these plants.

In a further embodiment the method comprises the steps a) growing theplants of the invention, b) removing the harvestable parts as definedabove from the plants and c) producing said product from or by theharvestable parts of the invention.

Preferably, the product obtained by said method comprises an exogenousACD nucleic acid, wherein the exogenous ACD nucleic acid is selectedfrom the group consisting of:

-   -   (i) an exogenous nucleic acid having at least 60%, preferably at        least 70%, for example at least 75%, more preferably at least        80%, for example at least 85%, even more preferably at least        90%, for example at least 95% or at least 96% or at least 97% or        at least 98% most preferably 99% identity with SEQ ID NO: 1,        3-10, 11, 13, 15, 17, 19, 21, 23, or 25, a functional fragment        thereof, or an orthologue or a paralogue thereof; or by    -   (ii) an exogenous nucleic acid encoding a protein having at        least 60% identity, preferably at least 70%, for example at        least 75%, more preferably at least 80%, for example at least        85%, even more preferably at least 90%, for example at least 95%        or at least 96% or at least 97% or at least 98% most preferably        99% homology with SEQ ID NO: 2, 12, 14, 16, 18, 20, 22, 24, or        26, a functional fragment thereof, an orthologue or a paralogue        thereof, or by    -   (iii) an exogenous nucleic acid capable of hybridizing under        stringent conditions with any of the nucleic acids according        to (i) or (ii) or a complementary sequence (complement) thereof,        and which preferably encodes a ACD protein that has essentially        the same biological activity as an ACD protein encoded by SEQ ID        NO: 2; preferably the encoded ACD protein confers enhanced        fungal resistance relative to control plants; or by    -   (iv) an exogenous nucleic acid encoding the same ACD protein as        the ACD nucleic acids of (i) to (iii) above, but differing from        the ACD nucleic acids of (i) to (iii) above due to the        degeneracy of the genetic code;        or wherein the product obtained by said method comprises an ACD        protein encoded by any one of the ACD nucleic acids of (i) to        (iv).

The product may be produced at the site where the plant has been grown,the plants and/or parts thereof may be removed from the site where theplants have been grown to produce the product. Typically, the plant isgrown, the desired harvestable parts are removed from the plant, iffeasible in repeated cycles, and the product made from the harvestableparts of the plant. The step of growing the plant may be performed onlyonce each time the methods of the invention is performed, while allowingrepeated times the steps of product production e.g. by repeated removalof harvestable parts of the plants of the invention and if necessaryfurther processing of these parts to arrive at the product. It is alsopossible that the step of growing the plants of the invention isrepeated and plants or harvestable parts are stored until the productionof the product is then performed once for the accumulated plants orplant parts. Also, the steps of growing the plants and producing theproduct may be performed with an overlap in time, even simultaneously toa large extend or sequentially. Generally the plants are grown for sometime before the product is produced.

In one embodiment the products produced by said methods of the inventionare plant products such as, but not limited to, a foodstuff, feedstuff,a food supplement, feed supplement, fiber, cosmetic and/orpharmaceutical. Foodstuffs are regarded as compositions used fornutrition and/or for supplementing nutrition. Animal feedstuffs andanimal feed supplements, in particular, are regarded as foodstuffs.

In another embodiment the inventive methods for the production are usedto make agricultural products such as, but not limited to, plantextracts, proteins, amino acids, carbohydrates, fats, oils, polymers,vitamins, and the like.

It is possible that a plant product consists of one or more agriculturalproducts to a large extent.

The transgenic plants of the invention may be crossed with similartransgenic plants or with transgenic plants lacking the nucleic acids ofthe invention or with non-transgenic plants, using known methods ofplant breeding, to prepare seeds. Further, the transgenic plant cells orplants of the present invention may comprise, and/or be crossed toanother transgenic plant that comprises one or more exogenous nucleicacids, thus creating a “stack” of transgenes in the plant and/or itsprogeny. The seed is then planted to obtain a crossed fertile transgenicplant comprising the ACD nucleic acid. The crossed fertile transgenicplant may have the particular expression cassette inherited through afemale parent or through a male parent. The second plant may be aninbred plant. The crossed fertile transgenic may be a hybrid. Alsoincluded within the present invention are seeds of any of these crossedfertile transgenic plants. The seeds of this invention can be harvestedfrom fertile transgenic plants and be used to grow progeny generationsof transformed plants of this invention including hybrid plant linescomprising the exogenous nucleic acid.

Thus, one embodiment of the present invention is a method for breeding afungal resistant plant comprising the steps of

-   (a) crossing a transgenic plant described herein or a plant    obtainable by a method described herein with a second plant;-   (b) obtaining a seed or seeds resulting from the crossing step    described in (a);-   (c) planting said seed or seeds and growing the seed or seeds to    plants; and-   (d) selecting from said plants the plants expressing a ACD protein,    preferably encoded by    -   (i) an exogenous nucleic acid having at least 60% identity with        SEQ ID NO: 1, 3-10, 11, 13, 15, 17, 19, 21, 23, or 25, a        functional fragment thereof, an orthologue or a paralogue        thereof;    -   (ii) an exogenous nucleic acid encoding a protein having at        least 60% identity with SEQ ID NO: 2, 12, 14, 16, 18, 20, 22,        24, or 26, or a functional fragment thereof, an orthologue or a        paralogue thereof;    -   (iii) an exogenous nucleic acid capable of hybridizing under        stringent conditions with any of the nucleic acids according        to (i) or (ii) or a complement thereof, and which preferably        encodes a ACD protein that has essentially the same biological        activity as an ACD protein encoded by SEQ ID NO: 2; preferably        the encoded ACD protein confers enhanced fungal resistance        relative to control plants; and/or by    -   (iv) an exogenous nucleic acid encoding the same ACD protein as        the ACD nucleic acids of (i) to (iii) above, but differing from        the ACD nucleic acids of (i) to (iii) above due to the        degeneracy of the genetic code.

The transgenic plants may be selected by known methods as describedabove (e.g., by screening for the presence of one or more markers whichare encoded by plant-expressible genes co-transferred with the ACD geneor screening for the ACD nucleic acid itself).

According to the present invention, the introduced ACD nucleic acid maybe maintained in the plant cell stably if it is incorporated into anon-chromosomal autonomous replicon or integrated into the plantchromosomes. Whether present in an extra-chromosomal non-replicating orreplicating vector construct or a vector construct that is integratedinto a chromosome, the exogenous ACD nucleic acid preferably resides ina plant expression cassette. A plant expression cassette preferablycontains regulatory sequences capable of driving gene expression inplant cells that are functional linked so that each sequence can fulfillits function, for example, termination of transcription bypolyadenylation signals. Preferred polyadenylation signals are thoseoriginating from Agrobacterium tumefaciens t-DNA such as the gene 3known as octopine synthase of the Ti-plasmid pTiACH5 (Gielen et al.,1984, EMBO J. 3:835) or functional equivalents thereof, but also allother terminators functionally active in plants are suitable. As plantgene expression is very often not limited on transcriptional levels, aplant expression cassette preferably contains other functional linkedsequences like translational enhancers such as the overdrive-sequencecontaining the 5′-untranslated leader sequence from tobacco mosaic virusincreasing the polypeptide per RNA ratio (Gallie et al., 1987, Nucl.Acids Research 15:8693-8711). Examples of plant expression vectorsinclude those detailed in: Becker, D. et al., 1992, New plant binaryvectors with selectable markers located proximal to the left border,Plant Mol. Biol. 20:1195-1197; Bevan, M. W., 1984, Binary Agrobacteriumvectors for plant transformation, Nucl. Acid. Res. 12:8711-8721; andVectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol.1, Engineering and Utilization, eds.: Kung and R. Wu, Academic Press,1993, S. 15-38.

FIGURES

FIG. 1 shows the schematic illustration of mode of action of the ACDprotein. The biosynthesis of ethylene in plants starts with theconversion of methionine to S-adenosyl-L-methionine (SAM) by SAMsynthetase (SAMS). In a second step SAM is converted to1-aminocyclopropane-1-carboxylic acid (ACC) by the enzyme ACC synthase(ACS). This step is the rate limiting step in the ethylene production inthe plant and therefore the tight regulation of this enzyme is key forethylene biosynthesis. In a final step ACC-oxidase (ACO) forms ethylenefrom ACC and oxygen. Binding of ET leads by the ethylene receptoractivates the ethylene signaling cascade leading to the expression of ETdependent genes.

FIG. 2 shows the scoring system used to determine the level of diseasedleaf area of wildtype and transgenic soy plants against the rust fungusP. pachyrhizi.

FIG. 3 shows the full-length-sequence of the ACD-gene from Pseudomonasspec. having SEQ ID NO: 1.

FIG. 4 shows the sequence of the ACD-protein (SEQ ID NO: 2).

FIG. 5 shows the result of the scoring of 13 transgenic soy plantsexpressing the ACD overexpression vector construct. To soybean plantsexpressing ACD protein were inoculated with spores of Phakopsorapachyrhizi. The evaluation of the diseased leaf area on all leaves wasperformed 14 days after inoculation. The average of the percentage ofthe leaf area showing fungal colonies or strong yellowing/browning onall leaves was considered as diseased leaf area. At all 13 soybean Toplants expressing ACD (expression checked by RT-PCR) were evaluated inparallel to non-transgenic control plants. The average of the diseasedleaf area is shown in FIG. 5. Overexpression of ACD reduces the diseasedleaf area in comparison to non-transgenic control plants by 42%.

FIG. 6 contains a brief description of the sequences of the sequencelisting.

EXAMPLES

The following examples are not intended to limit the scope of the claimsto the invention, but are rather intended to be exemplary of certainembodiments. Any variations in the exemplified methods that occur to theskilled artisan are intended to fall within the scope of the presentinvention.

Example 1 General Methods

The chemical synthesis of oligonucleotides can be affected, for example,in the known fashion using the phosphoamidite method (Voet, Voet, 2ndEdition, Wiley Press New York, pages 896-897). The cloning steps carriedout for the purposes of the present invention such as, for example,restriction cleavages, agarose gel electrophoresis, purification of DNAfragments, transfer of nucleic acids to nitrocellulose and nylonmembranes, linking DNA fragments, transformation of E. coli cells,bacterial cultures, phage multiplication and sequence analysis ofrecombinant DNA, are carried out as described by Sambrook et al. ColdSpring Harbor Laboratory Press (1989), ISBN 0-87969-309-6. Thesequencing of recombinant DNA molecules is carried out with an MWG-Licorlaser fluorescence DNA sequencer following the method of Sanger (Sangeret al., Proc. Natl. Acad. Sci. USA 74, 5463 (1977)).

Example 2 Cloning of Overexpression Vector Constructs

The cDNAs of all genes mentioned in this application were generated byDNA synthesis (Geneart, Regensburg, Germany).

The ACD cDNA were synthesized in a way that a attB1-recombination site(Gateway system, Invitrogen, Life Technologies, Carlsbad, Calif., USA)is located in front of the start-ATG and a attB2 recombination site islocated directly downstream of the stop-codon. The synthesized cDNA weretransferred to a pENTRY-B vector by using the BP reaction (Gatewaysystem, Invitrogen, Life Technologies, Carlsbad, Calif., USA) accordingto the protocol provided by the supplier. To obtain the binary planttransformation vector, a triple LR reaction (Gateway system, Invitrogen,Life Technologies, Carlsbad, Calif., USA) was performed according tomanufacturers protocol by using a pENTRY-A vector containing a parsleyubiquitine promoter, the cDNA in a pENTRY-B vector and a pENTRY-C vectorcontaining a Solanum tuberosum StCAT-pA terminator. As target a binarypDEST vector was used which is composed of: (1) aSpectinomycin/Streptomycin resistance cassette for bacterial selection(2) a pVS1 origin for replication in Agrobacteria (3) a pBR322 origin ofreplication for stable maintenance in E. coli and (4) between the rightand left border an AHAS selection under control of a pcUbi-promoter(FIG. 4). The recombination reaction was transformed into E. coli(DH5alpha), mini-prepped and screened by specific restrictiondigestions. A positive clone from each vector construct was sequencedand submitted soy transformation.

Example 3 Soy Transformation

The expression vector constructs (see example 2) were transformed intosoy.

3.1 Sterilization and Germination of Soy Seeds

Virtually any seed of any soy variety can be employed in the method ofthe invention. A variety of soycultivar (including Jack, Williams 82,Jake, Stoddard and Resnik) is appropriate for soy transformation. Soyseeds were sterilized in a chamber with a chlorine gas produced byadding 3.5 ml 12N HCl drop wise into 100 ml bleach (5.25% sodiumhypochlorite) in a desiccator with a tightly fitting lid. After 24 to 48hours in the chamber, seeds were removed and approximately 18 to 20seeds were plated on solid GM medium with or without 5 μM6-benzyl-aminopurine (BAP) in 100 mm Petri dishes. Seedlings without BAPare more elongated and roots develop, especially secondary and lateralroot formation. BAP strengthens the seedling by forming a shorter andstockier seedling.

Seven-day-old seedlings grown in the light (>100 μEinstein/m²s) at 25°C. were used for explant material for the three-explant types. At thistime, the seed coat was split, and the epicotyl with the unifoliateleaves have grown to, at minimum, the length of the cotyledons. Theepicotyl should be at least 0.5 cm to avoid the cotyledonary-node tissue(since soycultivars and seed lots may vary in the developmental time adescription of the germination stage is more accurate than a specificgermination time).

For inoculation of entire seedlings, see Method A (example 3.3. and3.3.2) or leaf explants, see Method B (example 3.3.3).

For method C (see example 3.3.4), the hypocotyl and one and a half orpart of both cotyledons were removed from each seedling. The seedlingswere then placed on propagation media for 2 to 4 weeks. The seedlingsproduce several branched shoots to obtain explants from. The majority ofthe explants originated from the plantlet growing from the apical bud.These explants were preferably used as target tissue.

3.2—Growth and Preparation of Agrobacterium Culture

Agrobacterium cultures were prepared by streaking Agrobacterium (e.g.,A. tumefaciens or A. rhizogenes) carrying the desired binary vector(e.g. H. Klee. R. Horsch and S. Rogers 1987 Agrobacterium-Mediated PlantTransformation and its further Applications to Plant Biology; AnnualReview of Plant Physiology Vol. 38: 467-486) onto solid YEP growthmedium YEP media: 10 g yeast extract, 10 g Bacto Peptone, 5 g NaCl,Adjust pH to 7.0, and bring final volume to 1 liter with H2O, for YEPagar plates add 20 g Agar, autoclave) and incubating at 25° C. untilcolonies appeared (about 2 days). Depending on the selectable markergenes present on the Ti or Ri plasmid, the binary vector, and thebacterial chromosomes, different selection compounds were be used for A.tumefaciens and rhizogenes selection in the YEP solid and liquid media.Various Agrobacterium strains can be used for the transformation method.

After approximately two days, a single colony (with a sterile toothpick)was picked and 50 ml of liquid YEP was inoculated with antibiotics andshaken at 175 rpm (25° C.) until an OD₆₀₀ between 0.8-1.0 is reached(approximately 2 d). Working glycerol stocks (15%) for transformationare prepared and one-ml of Agrobacterium stock aliquoted into 1.5 mlEppendorf tubes then stored at −80° C.

The day before explant inoculation, 200 ml of YEP were inoculated with 5μl to 3 ml of working Agrobacterium stock in a 500 ml Erlenmeyer flask.The flask was shaked overnight at 25° C. until the OD₆₀₀ was between 0.8and 1.0. Before preparing the soy explants, the Agrobacteria werepelleted by centrifugation for 10 min at 5,500×g at 20° C. The pelletwas resuspended in liquid CCM to the desired density (OD₆₀₀ 0.5-0.8) andplaced at room temperature at least 30 min before use.

3.3—Explant Preparation and Co-Cultivation (Inoculation)

3.3.1 Method A: Explant Preparation on the Day of Transformation.

Seedlings at this time had elongated epicotyls from at least 0.5 cm butgenerally between 0.5 and 2 cm. Elongated epicotyls up to 4 cm in lengthhad been successfully employed. Explants were then prepared with: i)with or without some roots, ii) with a partial, one or both cotyledons,all preformed leaves were removed including apical meristem, and thenode located at the first set of leaves was injured with several cutsusing a sharp scalpel.

This cutting at the node not only induced Agrobacterium infection butalso distributed the axillary meristem cells and damaged pre-formedshoots. After wounding and preparation, the explants were set aside in aPetri dish and subsequently co-cultivated with the liquidCCM/Agrobacterium mixture for 30 minutes. The explants were then removedfrom the liquid medium and plated on top of a sterile filter paper on15×100 mm Petri plates with solid co-cultivation medium. The woundedtarget tissues were placed such that they are in direct contact with themedium.

3.3.2 Modified Method A: Epicotyl Explant Preparation

Soyepicotyl segments prepared from 4 to 8 d old seedlings were used asexplants for regeneration and transformation. Seeds of soya cv.L00106CN, 93-41131 and Jack were germinated in 1/10 MS salts or asimilar composition medium with or without cytokinins for 4 to 8 d.Epicotyl explants were prepared by removing the cotyledonary node andstem node from the stem section. The epicotyl was cut into 2 to 5segments. Especially preferred are segments attached to the primary orhigher node comprising axillary meristematic tissue.

The explants were used for Agrobacterium infection. Agrobacterium AGL1harboring a plasmid with the gene of interest (GOI) and the AHAS, bar ordsdA selectable marker gene was cultured in LB medium with appropriateantibiotics overnight, harvested and resuspended in a inoculation mediumwith acetosyringone. Freshly prepared epicotyl segments were soaked inthe Agrobacterium suspension for 30 to 60 min and then the explants wereblotted dry on sterile filter papers. The inoculated explants were thencultured on a coculture medium with L-cysteine and TTD and otherchemicals such as acetosyringone for increasing T-DNA delivery for 2 to4 d. The infected epicotyl explants were then placed on a shootinduction medium with selection agents such as imazapyr (for AHAS gene),glufosinate (for bar gene), or D-serine (for dsdA gene). The regeneratedshoots were subcultured on elongation medium with the selective agent.

For regeneration of transgenic plants the segments were then cultured ona medium with cytokinins such as BAP, TDZ and/or Kinetin for shootinduction. After 4 to 8 weeks, the cultured tissues were transferred toa medium with lower concentration of cytokinin for shoot elongation.Elongated shoots were transferred to a medium with auxin for rooting andplant development. Multiple shoots were regenerated.

Many stable transformed sectors showing strong cDNA expression wererecovered. Soy-plants were regenerated from epicotyl explants. EfficientT-DNA delivery and stable transformed sectors were demonstrated.

3.3.3 Method B: Leaf Explants

For the preparation of the leaf explant the cotyledon was removed fromthe hypocotyl. The cotyledons were separated from one another and theepicotyl is removed. The primary leaves, which consist of the lamina,the petiole, and the stipules, were removed from the epicotyl bycarefully cutting at the base of the stipules such that the axillarymeristems were included on the explant. To wound the explant as well asto stimulate de novo shoot formation, any pre-formed shoots were removedand the area between the stipules was cut with a sharp scalpel 3 to 5times.

The explants are either completely immersed or the wounded petiole enddipped into the Agrobacterium suspension immediately after explantpreparation. After inoculation, the explants are blotted onto sterilefilter paper to remove excess Agrobacterium culture and place explantswith the wounded side in contact with a round 7 cm Whatman paperoverlaying the solid CCM medium (see above). This filter paper preventsA. tumefaciens overgrowth on the soy-explants. Wrap five plates withParafilm™ “M” (American National Can, Chicago, Ill., USA) and incubatefor three to five days in the dark or light at 25° C.

3.3.4 Method C: Propagated Axillary Meristem

For the preparation of the propagated axillary meristem explantpropagated 3-4 week-old plantlets were used. Axillary meristem explantscan be pre-pared from the first to the fourth node. An average of threeto four explants could be obtained from each seedling. The explants wereprepared from plantlets by cutting 0.5 to 1.0 cm below the axillary nodeon the internode and removing the petiole and leaf from the explant. Thetip where the axillary meristems lie was cut with a scalpel to induce denovo shoot growth and allow access of target cells to the Agrobacterium.Therefore, a 0.5 cm explant included the stem and a bud.

Once cut, the explants were immediately placed in the Agrobacteriumsuspension for 20 to 30 minutes. After inoculation, the explants wereblotted onto sterile filter paper to remove excess Agrobacterium culturethen placed almost completely immersed in solid CCM or on top of a round7 cm filter paper overlaying the solid CCM, depending on theAgrobacterium strain. This filter paper prevents Agrobacteriumovergrowth on the soy-explants. Plates were wrapped with Parafilm™ “M”(American National Can, Chicago, Ill., USA) and incubated for two tothree days in the dark at 25° C.

3.4—Shoot Induction

After 3 to 5 days co-cultivation in the dark at 25° C., the explantswere rinsed in liquid SIM medium (to remove excess Agrobacterium) (SIM,see Olhoft et al 2007 A novel Agrobacterium rhizogenes-mediatedtransformation method of soy using primary-node explants from seedlingsIn Vitro Cell. Dev. Biol.—Plant (2007) 43:536-549; to remove excessAgrobacterium) or Modwash medium (1× B5 major salts, 1× B5 minor salts,1× MSIII iron, 3% Sucrose, 1× B5 vitamins, 30 mM MES, 350 mg/L Timentin™pH 5.6, WO 2005/121345) and blotted dry on sterile filter paper (toprevent damage especially on the lamina) before placing on the solid SIMmedium. The approximately 5 explants (Method A) or 10 to 20 (Methods Band C) explants were placed such that the target tissue was in directcontact with the medium. During the first 2 weeks, the explants could becultured with or without selective medium. Preferably, explants weretransferred onto SIM without selection for one week.

For leaf explants (Method B), the explant should be placed into themedium such that it is perpendicular to the surface of the medium withthe petiole imbedded into the medium and the lamina out of the medium.

For propagated axillary meristem (Method C), the explant was placed intothe medium such that it was parallel to the surface of the medium(basipetal) with the explant partially embedded into the medium.

Wrap plates with Scotch 394 venting tape (3M, St. Paul, Minn., USA) wereplaced in a growth chamber for two weeks with a temperature averaging25° C. under 18 h light/6 h dark cycle at 70-100 μE/m²s. The explantsremained on the SIM medium with or without selection until de novo shootgrowth occurred at the target area (e.g., axillary meristems at thefirst node above the epicotyl). Transfers to fresh medium can occurduring this time. Explants were transferred from the SIM with or withoutselection to SIM with selection after about one week. At this time,there was considerable de novo shoot development at the base of thepetiole of the leaf explants in a variety of SIM (Method B), at theprimary node for seedling explants (Method A), and at the axillary nodesof propagated explants (Method C).

Preferably, all shoots formed before transformation were removed up to 2weeks after co-cultivation to stimulate new growth from the meristems.This helped to reduce chimerism in the primary transformant and increaseamplification of transgenic meristematic cells. During this time theexplant may or may not be cut into smaller pieces (i.e. detaching thenode from the explant by cutting the epicotyl).

3.5—Shoot Elongation

After 2 to 4 weeks (or until a mass of shoots was formed) on SIM medium(preferably with selection), the explants were transferred to SEM medium(shoot elongation medium, see Olhoft et al 2007 A novel Agrobacteriumrhizogenes-mediated transformation method of soy using primary-nodeexplants from seedlings. In Vitro Cell. Dev. Biol.—Plant (2007)43:536-549) that stimulates shoot elongation of the shoot primordia.This medium may or may not contain a selection compound.

After every 2 to 3 weeks, the explants were transfer to fresh SEM medium(preferably containing selection) after carefully removing dead tissue.The explants should hold together and not fragment into pieces andretain somewhat healthy. The explants were continued to be transferreduntil the explant dies or shoots elongate. Elongated shoots>3 cm wereremoved and placed into RM medium for about 1 week (Method A and B), orabout 2 to 4 weeks depending on the cultivar (Method C) at which timeroots began to form. In the case of explants with roots, they weretransferred directly into soil. Rooted shoots were transferred to soiland hardened in a growth chamber for 2 to 3 weeks before transferring tothe greenhouse. Regenerated plants obtained using this method werefertile and produced on average 500 seeds per plant.

After 5 days of co-cultivation with Agrobacterium tumefaciens transientexpression of the gene of interest (GOI) was widespread on the seedlingaxillary meristem explants especially in the regions wounding duringexplant preparation (Method A). Explants were placed into shootinduction medium without selection to see how the primary-node respondsto shoot induction and regeneration. Thus far, greater than 70% of theexplants were formed new shoots at this region. Expression of the GOIwas stable after 14 days on SIM, implying integration of the T-DNA intothe soy genome. In addition, preliminary experiments resulted in theformation of cDNA expressing shoots forming after 3 weeks on SIM.

For Method C, the average regeneration time of a soy plantlet using thepropagated axillary meristem protocol was 14 weeks from explantinoculation. Therefore, this method has a quick regeneration time thatleads to fertile, healthy soy plants.

Example 4 Pathogen Assay

4.1. Recovery of Clones

2-3 clones per T₀ event were potted into small 6 cm pots. For recoverythe clones were kept for 12-18 days in the phytochamber (16 h-day- and 8h-night-Rhythm at a temperature of 16°-22° C. and a humidity of 75%).

4.2 Inoculation

The plants were inoculated with P. pachyrhizi

In order to obtain appropriate spore material for the inoculation, soyleaves which had been infected with rust 15-20 days ago, were taken 2-3days before the inoculation and transferred to agar plates (1% agar inH2O). The leaves were placed with their upper side onto the agar, whichallowed the fungus to grow through the tissue and to produce very youngspores. For the inoculation solution, the spores were knocked off theleaves and were added to a Tween-H2O solution. The counting of sporeswas performed under a light microscope by means of a Thoma countingchamber. For the inoculation of the plants, the spore suspension wasadded into a compressed-air operated spray flask and applied uniformlyonto the plants or the leaves until the leaf surface is wellmoisturized. For macroscopic assays we used a spore density of 1-5×105spores/ml. For the microscopy, a density of >5×105 spores/ml is used.The inoculated plants were placed for 24 hours in a greenhouse chamberwith an average of 22° C. and >90% of air humidity. The followingcultivation was performed in a chamber with an average of 25° C. and 70%of air humidity.

Example 5 Microscopical Screening

For the evaluation of the pathogen development, the inoculated leaves ofplants were stained with aniline blue 48 hours after infection.

The aniline blue staining serves for the detection of fluorescentsubstances. During the defense reactions in host interactions andnon-host interactions, substances such as phenols, callose or ligninaccumulated or were produced and were incorporated at the cell walleither locally in papillae or in the whole cell (hypersensitivereaction, HR). Complexes were formed in association with aniline blue,which lead e.g. in the case of callose to yellow fluorescence. The leafmaterial was transferred to falcon tubes or dishes containing destainingsolution II (ethanol/acetic acid 6/1) and was incubated in a water bathat 90° C. for 10-15 minutes. The destaining solution II was removedimmediately thereafter, and the leaves were washed 2× with water. Forthe staining, the leaves were incubated for 1.5-2 hours in stainingsolution II (0.05% aniline blue=methyl blue, 0.067 M di-potassiumhydrogen phosphate) and analyzed by microscopy immediately thereafter.

The different interaction types were evaluated (counted) by microscopy.An Olympus UV microscope BX61 (incident light) and a UV Longpath filter(excitation: 375/15, Beam splitter: 405 LP) are used. After aniline bluestaining, the spores appeared blue under UV light. The papillae could berecognized beneath the fungal appressorium by a green/yellow staining.The hypersensitive reaction (HR) was characterized by a whole cellfluorescence.

Example 6 Evaluating the Susceptibility to Soybean Rust

The progression of the soybean rust disease was scored by the estimationof the diseased area (area which was covered by sporulating uredinia) onthe backside (abaxial side) of the leaf. Additionally the yellowing ofthe leaf was taken into account (for scheme see FIG. 2).

To soybean plants expressing ACD protein were inoculated with spores ofPhakopsora pachyrhizi. The macroscopic disease symptoms of soy againstP. pachyrhizi of 13 To soybean plants were scored 14 days afterinoculation.

The average of the percentage of the leaf area showing fungal coloniesor strong yellowing/browning on all leaves was considered as diseasedleaf area. At all 13 soybean To plants expressing ACD (expressionchecked by RT-PCR) were evaluated in parallel to non-transgenic controlplants. Clones from non-transgenic soy plants were used as control. Theaverage of the diseased leaf area is shown in FIG. 5 for plantsexpressing recombinant ACD compared with wildtype plants. Overexpressionof ACD reduces the diseased leaf area in comparison to non-transgeniccontrol plants by 42% in average over all events generated. This dataclearly indicate that the in planta expression of the ACD expressionvector construct lead to a lower disease scoring of transgenic plantscompared to non-transgenic controls. So, the expression of ACD in soyincreases the resistance of soy against soybean rust.

The invention claimed is:
 1. A method for increasing resistance againstPhakopsora in a plant, a plant part, or a plant cell, the methodcomprising the step of increasing the expression and/or activity of anaminocyclopropane carboxylic acid deaminase (ACD) protein in the plant,plant part, or plant cell in comparison to a wild type plant, wild typeplant part or wild type plant cell, wherein the ACD protein is encodedby an exogenous nucleic acid encoding a protein having an amino acidsequence with at least 90% identity to SEQ ID NO:
 2. 2. The method ofclaim 1, comprising: (a) stably transforming a plant cell with anexpression cassette comprising: an exogenous nucleic acid encoding aprotein having an amino acid sequence with at least 90% identity to SEQID NO: 2 in functional linkage with a promoter; (b) regenerating theplant from the plant cell; and (c) expressing said exogenous nucleicacid.
 3. The method of claim 2, wherein the promoter is a constitutive,pathogen-inducible promoter, a mesophyll-specific promoter or anepidermis specific-promoter.
 4. The method of claim 1, wherein theresistance against Phakopsora is resistance against Phakopsorameibomiae, Phakopsora pachyrhizi, or combinations thereof.
 5. The methodof claim 1, wherein the plant is selected from the group consisting ofbeans, soya, pea, clover, kudzu, lucerne, lentils, lupins, vetches,groundnut, rice, wheat, barley, arabidopsis, lentil, banana, canola,cotton, potatoe, corn, sugar cane, alfalfa, and sugar beet.